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Abstract Background Surfactant protein D (SP-D) plays an important role in innate defense against influenza A viruses (IAVs) and other pathogens. Methods We tested antiviral activities of recombinant human SP-D against a panel of IAV strains that vary in glycosylation sites on their hemagglutinin (HA). For these experiments a recombinant version of human SP-D of the Met11, Ala160 genotype was used after it was characterized biochemically and structurally. Results Oligosaccharides at amino acid 165 on the HA in the H3N2 subtype and 104 in the H1N1 subtype are absent in collectin-resistant strains developed in vitro and are important for mediating antiviral activity of SP-D; however, other glycans on the HA of these viral subtypes also are involved in inhibition by SP-D. H3N2 strains obtained shortly after introduction into the human population were largely resistant to SP-D, despite having the glycan at 165. H3N2 strains have become steadily more sensitive to SP-D over time in the human population, in association with addition of other glycans to the head region of the HA. In contrast, H1N1 strains were most sensitive in the 1970s–1980s and more recent strains have become less sensitive, despite retaining the glycan at 104. Two H5N1 strains were also resistant to inhibition by SP-D. By comparing sites of glycan attachment on sensitive vs. resistant strains, specific glycan sites on the head domain of the HA are implicated as important for inhibition by SP-D. Molecular modeling of the glycan attachment sites on HA and the carbohydrate recognition domain of SPD are consistent with these observations. Conclusion Inhibition by SP-D correlates with presence of several glycan attachment sites on the HA. Pandemic and avian strains appear to lack susceptibility to SP-D and this could be a contributory factor to their virulence.

Abstract Background Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection. Common human polymorphisms of SP-D have been found in many human populations and associated with increased risk of certain infections. We recently reported that the Thr/Thr 11 form of SP-D is associated with low serum levels and assembles predominantly as trimers as opposed to the more common multimeric forms of SP-D. Methods Preliminary experiments were done to establish the effects of different monoclonal antibodies against SP-D on ability of SP-D to bind to or neutralize the virus. We then purified natural human trimeric and multimeric forms of SP-D from amniotic fluid and tested ability of these preparations to bind to IAV, to inhibit infectivity and hemagglutination activity of IAV in vitro. Results In initial experiments mAbs directed against different areas on the CRD of SP-D were found to have differing effects on antiviral activity. Using an mAb that did not interfere with antiviral activity of SP-D, we confirm that natural SP-D trimers had reduced ability to bind to IAV. In addition, the trimers had reduced ability to neutralize IAV as compared to natural human SP-D multimers as well as reduced hemagglutination inhibiting activity against several strains of IAV. Natural SP-D trimers also had different interactions with human neutrophil peptide defensins (HNPs) in viral neutralization assays as compared to multimeric SP-D. Conclusion These studies indicate that a common human polymorphic form of SP-D may modulate host defense against IAV and give impetus to clinical studies correlating this genotype with risk for IAV infection in susceptible groups. We also show that mAbs directed against different areas on the carbohydrate recognition domain of SP-D can be useful for dissecting out different functional properties of the protein.

Increased interest in greenhouse gas (GHG) emissions, including recent legislative action and voluntary programs, has increased attention on quantifying and ultimately reducing methane emissions from the natural gas supply chain. While inventories used for public or corporate GHG policies have traditionally utilized bottom-up (BU) methods to estimate emissions, the validity of such inventories has been questioned. Therefore, there is attention on utilizing full-facility measurements using airborne, satellite, or drone (top-down (TD)) techniques to inform, improve, or validate inventories. This study utilized full-facility estimates from two independent TD methods at 15 midstream natural gas facilities in the U.S.A., which were compared with a contemporaneous daily inventory assembled by the facility operator, employing comprehensive inventory methods. Estimates from the two TD methods statistically agreed in 2 of 28 paired measurements. Operator inventories, which included extensions to capture sources beyond regular inventory requirements and integration of local measurements, estimated significantly lower emissions than the TD estimates for 40 of 43 paired comparisons. Significant disagreement was observed at most facilities, both between the two TD methods and between the TD estimates and operator inventory. These findings have two implications. First, improving inventory estimates will require additional on-site or ground-based diagnostic screening and measurement of all sources. Second, the TD full-facility measurement methods need to undergo further testing, characterization, and potential improvement specifically tailored for complex midstream facilities.

The status and habitat selection of the three species of gazelle indigenous to Eritrea, i.e., Nanger soemmerringii, Gazella dorcas and Eudorcas tilonura, are not well known. In this study, we analyzed the present distribution of the three species in the country in order to identify preferred habitats and assess the effect of human disturbance (land use for agricultural purposes and livestock) on species occurrence. These data represent baseline information for evidence-based strategies for conservation of the three species in Eritrea. Presence/absence data of the three species in each of the 67 administrative subregions (Sub Zoba) composing the country were collected using direct (field surveys) and indirect methods (questionnaires). For each sampling unit, we collected fifteen environmental variables, of which three are associated with climatic features, eight with vegetation structure and four with human disturbance (human-related land use and livestock). The occurrence probability of each species was modeled through Generalized Linear Models (GLM). The analyses showed that Dorcas gazelle occurred more frequently in warmer conditions and in a wide range of natural vegetation types. Heuglin's gazelle occurred in warmer regions with higher seasonality in both temperature and precipitation with a preference for closed woody and open grassland areas. In the case of Soemmerring's gazelle, the GLM with climatic variables predicted a preference for warmer conditions but with lower seasonality of temperature and precipitation. The species also seemed to prefer arid and semi-arid open vegetation. Human disturbance is the variable with the strongest, negative, effect on the species occurrence. Indeed, the occurrence probability of each species decreased with increasing livestock density and agricultural land use. Most of these gazelle occurred in unprotected areas, thus the human-related activities are undoubtedly the most important threat for the three species of gazelle in Eritrea. Therefore, the establishment of protected areas that preserve the potential optimal habitats for gazelle and reduce the impact of livestock ranching are essential to ensure a future for these gazelle in Eritrea.

The industrial sector represents roughly 22% of U.S. emissions. Unlike emissions from fossil-fueled power plants, the carbon footprint of the industrial sector represents a complex mixture of stationary combustion and process emissions produced as a reaction byproduct of cement, iron and steel, glass, and oil production. This study quantifies the potential opportunities for low-cost carbon capture and storage (CCS) scenarios with process emissions from the U.S. industrial sector by analyzing the variabilities in point-source capture and geographic proximity to relevant sinks, specifically enhanced oil recovery (EOR) and geologic sequestration opportunities. Using a technology-agnostic cost model developed from mature CO 2 capture technologies, costs of CCS are calculated for each of the 656 facilities considered, with application of the U.S. federal tax credit 45Q to qualifying facilities. Capture of these targeted industrial process emission streams may lead to the avoidance of roughly 195 MtCO 2 /yr (188 MtCO 2 /yr qualifying for 45Q). A total of 123 facilities have the potential to avoid roughly 68.5 MtCO 2 /yr at costs below $40/tCO 2 delivered. This could be competitive for using CO 2 for EOR depending on the price of oil. At regional CO 2 collection hubs, emissions of 40 MtCO 2 /yr can be avoided within 100 miles of the existing Louisiana-Mississippi and Texas-New Mexico pipelines.

LL-37, the only human cathelicidin, is a cationic antimicrobial peptide with antibacterial and antifungal activity. LL-37 is released from neutrophil granules and produced by epithelial cells. It has been implicated in host defence against influenza A virus (IAV) in recent studies. We now demonstrate dose-related neutralizing activity of LL-37 against several seasonal and mouse-adapted IAV strains. The ability of LL-37 to inhibit these IAV strains resulted mainly from direct effects on the virus, since pre-incubation of virus with LL-37 was needed for optimal inhibition. LL-37 bound high-density lipoprotein (HDL), and pre-incubation of LL-37 with human serum or HDL reduced its antiviral activity. LL-37 did not inhibit viral association with epithelial cells as assessed by quantitative RT-PCR or confocal microscopy. This finding contrasted with results obtained with surfactant protein D (SP-D). Unlike collectins or human neutrophil defensins (HNPs), LL-37 did not induce viral aggregation under electron microscopy. In the electron microscopy studies, LL-37 appeared to cause disruption of viral membranes. LL-37 had additive antiviral activity when combined with other innate inhibitors like SP-D, surfactant protein A and HNPs. Unlike HNPs, LL-37 did not bind SP-D significantly. These findings indicate that LL-37 contributes to host defence against IAV through a mechanism distinct from that of SP-D and HNPs.

θ-Defensins are cyclic octadecapeptides found in nonhuman primates whose broad antiviral spectrum includes HIV-1, HSV-1, severe acute respiratory syndrome coronavirus, and influenza A virus (IAV). We previously reported that synthetic θ-defensins called retrocyclins can neutralize and aggregate various strains of IAV and increase IAV uptake by neutrophils. This study describes two families of peptides, hapivirins and diprovirins, whose design was inspired by retrocyclins. The goal was to develop smaller partially cyclic peptides that retain the antiviral activity of retrocyclins, while being easier to synthesize. The novel peptides also allowed for systemic substitution of key residues to evaluate the role of charge or hydrophobicity on antiviral activity. Seventy-two hapivirin or diprovirin peptides are described in this work, including several whose anti-IAV activity equals or exceeds that of normal α- or θ-defensins. Some of these also had strong antibacterial and antifungal activity. These new peptides were active against H3N2 and H1N1 strains of IAV. Structural features imparting strong antiviral activity were identified through iterative cycles of synthesis and testing. Our findings show the importance of hydrophobic residues for antiviral activity and show that pegylation, which often increases a peptide's serum t(1/2) in vivo, can increase the antiviral activity of DpVs. The new peptides acted at an early phase of viral infection, and, when combined with pulmonary surfactant protein D, their antiviral effects were additive. The peptides strongly increased neutrophil and macrophage uptake of IAV, while inhibiting monocyte cytokine generation. Development of modified θ-defensin analogs provides an approach for creating novel antiviral agents for IAV infections.

Defensins were first identified in 1985 and are now recognized as part of a large family of antimicrobial peptides, divided into three categories: alpha-, beta-, and -defensins. These defensin classes differ in structure, sites of expression and biological activities. Human alpha-defensins include peptides that are expressed primarily in neutrophils, whereas human beta-defensins are widely expressed in epithelial cells, including those lining the respiratory tract. Defensins were first studied for their broad spectrum activity against bacteria, fungi and viruses; however, it is now clear that they also recruit inflammatory cells and promote innate and adaptive immune responses. Recent evidence shows that defensins have anti-inflammatory effects as well. Hence, defensins can participate in all phases of an immune response in the lung, including initial killing of pathogens and mounting - and resolution -- of an immune or inflammatory response. The cathelicidin, LL-37, is an antimicrobial peptide produced by neutrophils and respiratory epithelial cells that has similar roles in lung immunity as the defensins. A major challenge for the coming years will be to sort out the relative contributions of defensins and LL-37 to overall immune responses in the lung and to determine which of their many in vitro activities are most important for lung immunity.

Collectins are collagenous lectins present in blood, respiratory lining fluid, and other mucosal secretions that play important roles in innate defense against infection. The collectin, surfactant protein D (SP-D), limits infection by viruses and bacteria in the respiratory tract, eye, and female genital tract. Multimeric SP-D has strong antiviral activity and is a potent viral and bacterial agglutinin and opsonin; however, trimers composed of the neck and carbohydrate recognition domain (hSP-D-NCRD) of SP-D lack these activities. We now show that, in contrast, a trimeric neck and CRD construct of bovine serum collectin CL-46 induces aggregation of influenza A virus (IAV) and potently increases IAV uptake by neutrophils. CL-46-NCRD showed calcium-dependent and sugar-sensitive binding to both neutrophils and IAV. Replacement of specific residues of the CRD of human SP-D with those found in bovine serum collectins conferred opsonizing activity. The most effective substitution involved replacement of arginine 343 with valine (hSP-D-NCRD/R343V). hSP-D-NCRD/R343V greatly increased viral uptake by neutrophils and monocytes and also potentiated neutrophil respiratory burst responses. These effects were further increased by cross-linking of hSP-D-NCRD/R343V trimers with MAbs directed against areas of the hSP-D-NCRD not involved in viral binding. Unlike the wild-type human SP-D hSP-D-NCRD, hSP-D-NCRD/R343V also induced viral aggregation. These results indicate that collectins can act as opsonins for IAV even in the absence of the collagen domain or higher order multimerization. This may involve increased affinity of individual CRDs for glycoconjugates displayed on host cells or the viral envelope.

Defensins are widespread in nature and have activity against a broad range of pathogens. Defensins have direct antimicrobial effects and also modulate innate and adaptive immune responses. We consider the role of human defensins and the cathelicidin LL-37 in defense of respiratory, gastrointestinal, and genitourinary tracts and the oral cavity, skin, and eye. Human beta-defensins (hBDs) and human defensins 5 and 6 (HD5 and -6) are involved most obviously in mucosal responses, as they are produced principally by epithelial cells. Human alpha-defensins 1-4 (or HNPs 1-4) are produced principally by neutrophils recruited to the mucosa. Understanding the biology of defensins and LL-37 is the beginning to clarify the pathophysiology of mucosal inflammatory and infectious diseases (e.g., Crohn's disease, atopic dermatitis, lung or urinary infections). Challenges for these studies are the redundancy of innate defense mechanisms and the presence and interactions of many innate defense proteins in mucosal secretions.

Surfactant protein D (SP-D) plays important roles in innate defense against respiratory viruses [including influenza A viruses (IAVs)]. Truncated trimers composed of its neck and carbohydrate recognition domains (NCRDs) bind various ligands; however, they have minimal inhibitory activity for IAV. We have sought to find ways to increase the antiviral activity of collectin NCRDs. Cross-linking of the SP-D NCRD with nonblocking monoclonal antibodies (mAbs) markedly potentiates antiviral activity. In the present report, we demonstrate that F(ab')2 [but not F(ab')1] fragments of a cross-linking mAb have similar effects. Hence, cross-linking activity, but not the Fc domain of the mAb, is needed for increased antiviral activity. In contrast, the Fc domain of the mAb was important for increasing viral uptake or respiratory burst responses of human neutrophils. Our NCRD constructs contain an S protein binding site. Herein, we show that a multivalent S protein complex caused cross-linking and also increased the antiviral activity of NCRDs. NCRDs of conglutinin and CL43 had greater intrinsic antiviral activity than those of SP-D or mannose-binding lectin. Based on motifs found in these serum collectins, we have constructed mutant versions of the human SP-D NCRD that have increased antiviral activity. These mutant NCRDs also had potentiated activity after cross-linking with F(ab')2 fragments or S protein complexes. Hence, the antiviral activity of NCRDs can be increased by 2 distinct, complementary strategies, namely cross-linking of NCRDs through various means and mutagenesis of CRD residues to increase viral binding. These findings may be relevant for antiviral therapy., ((c) 2009 S. Karger AG, Basel.)

We have reported that the alpha-defensins human neutrophil peptides (HNP)-1 and HNP-2 neutralize and aggregate influenza A virus (IAV) and promote uptake of IAV by neutrophils. These alpha-defensins were also shown to bind to surfactant protein (SP)-D and reduce its antiviral activity. In this study, we examined retrocyclin (RC)1 and RC2, humanized versions of the antiviral theta-defensins found in the leukocytes of certain nonhuman primates. RC1 was just as effective as HNP-1-3 in neutralizing IAV, and RC2 and RC101 (an analog of RC1) were more effective. In contrast, human beta-defensins (HBDs) showed less neutralizing activity. Human defensins 5 and 6 (mainly produced by intestinal Paneth cells) had viral neutralizing activity similar to HNP-1-3. Like HNP-1-3, RCs induced viral aggregation and promoted the uptake of IAV by neutrophils. We used surface plasmon resonance to evaluate binding of defensins to SP-D. HBDs, HD6, and HNP-4 bound minimally to SP-D. HNP-1-3 and RCs bound SP-D with high affinity; however, unlike HNP-1 and HNP-2, RCs did not inhibit SP-D antiviral activity. HBDs also did not inhibit antiviral activity of SP-D. Given their strong neutralizing activity and compatibility with SP-D, RCs may provide attractive prototypes for designing therapeutics that can prevent or treat respiratory infections caused by IAV.

Surfactant protein D (SP-D) plays important roles in the initial innate defense against influenza A virus (IAV). The collagen domain of SP-D is probably critical for its homeostatic functions in vivo and has been implicated in the modulation of macrophage responses to SP-D-ligand complexes. For the current studies, we used a panel of rat SP-D mutants lacking all or part of the collagen domain to more specifically evaluate the contributions of this domain to viral interactions. SP-D multimers lacking the collagenous sequence efficiently neutralized Phil82 IAV, promoted neutrophil uptake of IAV, and also potentiated the IAV-induced neutrophil respiratory burst response. A dodecameric mutant with shortened collagenous arms showed enhanced viral aggregation and neuraminidase inhibition, and an increased capacity to inhibit a partially collectin-resistant strain of IAV. By contrast, truncated molecules lacking an N-terminal and collagen domain showed no detectable antiviral and opsonizing activity, despite preservation of lectin activity and detectable viral binding. Thus, multimerization, which is mediated by the N-peptide, is more important than the collagen domain for efficient viral neutralization and opsonization. However, the structure of the collagen domain significantly influences the anti-viral activity of multimerized forms of SP-D.

Innate immunity is critical in the early containment of influenza virus infection. The innate response is surprisingly complex. A variety of soluble innate inhibitors in respiratory secretions provide an initial barrier to infection. Dendritic cells, phagocytes and natural killer cells mediate viral clearance and promote further innate and adaptive responses. Toll-like receptors 3 and 7 and cytoplasmic RNA sensors are critical for activating these responses. In general, the innate response restricts viral replication without injuring the lung; however, the 1918 pandemic and H5N1 strains cause more profound, possibly harmful, innate responses. In this review, we discuss the implications of burgeoning knowledge of innate immunity for therapy of influenza.

Collectins are multimeric host defence lectins with trimeric CRDs (carbohydrate-recognition domains) and collagen and N-terminal domains that form higher-order structures composed of four or more trimers. Recombinant trimers composed of only the CRD and adjacent neck domain (termed NCRD) retain binding activity for some ligands and mediate some functional activities. The lung collectin SP-D (surfactant protein D) has strong neutralizing activity for IAVs (influenza A viruses) in vitro and in vivo, however, the NCRD derived from SP-D has weak viral-binding ability and lacks neutralizing activity. Using a panel of mAbs (monoclonal antibodies) directed against the NCRD in the present study we show that mAbs binding near the lectin site inhibit antiviral activity of full-length SP-D, but mAbs which bind other sites on the CRD do not. Two of the non-blocking mAbs significantly increased binding and antiviral activity of NCRDs as assessed by haemagglutination and neuraminidase inhibition and by viral neutralization. mAb-mediated cross-linking also enabled NCRDs to induce viral aggregation and to increase viral uptake by neutrophils and virus-induced respiratory burst responses by these cells. These results show that antiviral activities of SP-D can be reproduced without the N-terminal and collagen domains and that cross-linking of NCRDs is essential for antiviral activity of SP-D with respect to IAV.

Surfactant protein D (SP-D) and neutrophils participate in the early innate immune response to influenza A virus (IAV) infection. SP-D increases neutrophil uptake of IAV and modulates neutrophil respiratory burst responses to IAV; however, neutrophil proteases have been shown to degrade SP-D, and human neutrophil peptide defensins bind to SP-D and can cause precipitation of SP-D from bronchoalveolar lavage fluid (BALF). BALF has significant antiviral activity against IAV. We first added neutrophils to BALF during incubation with IAV. Addition of neutrophils to BALF caused significantly greater clearance of IAV from culture supernatants than from BALF alone, and this effect was significantly more pronounced when neutrophils were activated during incubation with the virus. In contrast, if activated neutrophils were incubated with BALF before addition of virus, they reduced antiviral activity of BALF. This effect correlated with depletion of SP-D from BALF. Activation of neutrophils with agonists that induce primary granule release (including release of human neutrophil peptide defensins) caused SP-D depletion, but activation with PMA, which causes only secondary granule release, did not. The ability of activated neutrophils to deplete SP-D from BALF was partially, but not fully, corrected with protease inhibitors but was unaffected by inhibition of neutrophil respiratory burst responses. These results suggest that chronic neutrophilic inflammation (e.g., as in chronic smoking or cystic fibrosis) may reduce SP-D levels and predispose to IAV infection. In contrast, acute inflammation, as occurs in the early phase of IAV infection, may promote neutrophil-mediated viral clearance.

Human neutrophil peptides (HNPs) are released from granules of neutrophils in response to various activating stimuli and they participate in the killing of bacteria and the stimulation of various inflammatory responses. HNPs also inhibit infectivity of enveloped viruses, including influenza A virus (IAV). In this study, we demonstrate that HNPs increase the uptake of IAV and bacteria by neutrophils. The dimeric HNPs also induced aggregation of IAV and bacterial particles, which may, in part, explain their ability to increase uptake. HNPs did not increase neutrophil respiratory burst responses to IAV. We have recently demonstrated direct interactions of HNPs with surfactant protein D (SP-D), another important effector of innate immunity and antimicrobial host defense. Although HNPs did not alter SP-D-dependent uptake of IAV, they counteracted the ability of SP-D to increase IAV-induced neutrophil H2O2 generation. Our studies reveal previously unappreciated functional effects of HNPs, expand our understanding of the antiviral properties of HNPs, and suggest important interactions between collectins and HNPs in the host response to viruses and bacteria.

Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some instances when relatively SP-D-resistant strains were treated with SP-D and HNPs. HNPs were found to bind to the neck and/or carbohydrate recognition domain of SP-D. This binding was specific because no, or minimal, binding to other collectins was found. HNPs precipitated SP-D from bronchoalveolar lavage fluid and reduced the antiviral activity of bronchoalveolar lavage fluid. HNP-1 and -2 differed somewhat in their independent antiviral activity and their binding to SP-D. These results are relevant to the early phase of host defense against IAV, and suggest a complex interplay between SP-D and HNPs at sites of active inflammation.

The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate-binding activity and was dependent on glycosylation of gp120. Native dodecameric SP-D bound to HIV gp120 more strongly than native trimeric SP-D. Since one common polymorphic form of SP-D is predominantly expressed as trimers and associated with lower blood levels, these individuals may have less effective innate defence against HIV. A chimeric protein containing the N-terminal and collagen domains of SP-D linked to the neck and carbohydrate-recognition domains of MBL (called SP-D/MBL(neck+CRD)) had greater ability to bind to gp120 and inhibit virus replication than either SP-D or MBL. The enhanced binding of SP-D/MBL(neck+CRD) was dependent on assembly into higher molecular mass multimers (i.e. a trimeric form of the chimera did not bind to a greater extent than MBL). Hence, the enhanced binding of SP-D compared with MBL results from distinctive properties of its N-terminal and/or collagen domains. SP-D is present in lung and airway fluids, as well as in blood and various mucosal locations, and could, like MBL, play a role in restricting HIV transmission or replication in vivo.

Mumps virus strains isolated during an epidemic in Lithuania in 1998 - 2000 were studied. Viruses of the neurovirulent C1 and non-neurovirulent C2 small hydrophobic (SH) genotype variant were sequenced for the haemagglutinin-neuraminidase (HN) and fusion (F) protein genes. Amino acid differences between C1 and C2 strains were found for both proteins. Two amino acid differences were of potential importance for the non-neurovirulent phenotype of the C2 virus. Four of 5 C2 strains exhibited the amino acid arginine instead of lysine at position 335 of the HN protein, and the amino acid phenylalanine was found instead of serine at amino acid position 195 of the F protein. Amino acid differences at these positions have previously been reported to associate with a change in neurovirulence and fusion activity. In addition, the HN gene of the neurovirulent Kilham strain of genotype A was sequenced. The deduced amino acid sequence showed different amino acids compared to both genotypes A and C on some positions. Notably, amino acid differences located in previously identified neutralizing epitopes were found at positions 266, 354 and 356 of the HN protein compared to other genotype A strains. The amino acid differences between Kilham virus strain and other genotype A strains and the similarity of the Kilham HN protein (7 positions) to neurovirulent genotype C strains on some amino acid positions may indicate a possible role for this protein in mumps virus neurovirulence.

The European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), is a major pest of maize, Zea mays L., in many temperate parts of the world. Genotype-by-environment interaction effects can make relative performance unpredictable and may hamper selection for resistance to European corn borer. The objective of this study was to determine the effect of environment on genotypic reaction to European corn borer resistance in maize. A set of 12 maize inbred lines was chosen to represent a range of European corn borer responses. Eleven testing environments ranged from Delaware, Ohio, Illinois, Iowa, Nebraska, Missouri, to Mississippi. For length of stalk tunneling, environmental and genotypic main effects (estimated by restricted maximum likelihood) were >20- and 10-fold larger than their interaction effect, respectively. Length of tunneling means for genotypes (across environments) ranged from 10.1 to 35.4 cm. Several putatively resistant genotypes grouped with the susceptible checks, B73 and Mol7. By breaking factors and the interaction into single degree of freedom components, we observed that GEMS-0001 had significant crossover interactions toward less susceptibility in both Mississippi and the Nebraska environments. Environments displaying several crossover interactions indicated that European corn borer screening at these sites would not necessarily apply to other locations, whether due to small differences in experimental conduct and/or environmental effects. The five most resistant genotypes were fairly consistent across environments. Because all environments except Illinois used larvae from the same insectary, and these environments differed in damage intensity and rankings, it is unlikely that insect biotype was a factor contributing to genotype-by-environment effects.

The epidemiology of respiratory syncytial virus (RSV) group A was followed by nucleotide sequencing of the variable parts of the glycoprotein (G) gene. The amino acid sequences of an aminoterminal (A-terminal, amino acids 90-132) and carboxyterminal (C-terminal, amino acids 262-298) portion of the G protein in 47 virus strains, collected in Stockholm, between 1965 and 2004, were determined. In phylogenetic analysis jointly with previously described genotypes (GA1 to GA7 and SAA1), 33 virus strains (isolated between 1991 and 2004) belonged to genotype GA5, seven to GA2, three to genotype GA1 (isolated before 1991), one to genotype GA4 (isolated in 1982) and three to genotype GA7 (isolated in 1993 and 2001). Genotype GA5 was predominant in four epidemics, between 2000/2001 and 2003/2004. Little or no variation with time of the C-terminal amino acid sequence of the G protein was found when the virus strains were compared within their own genotype. Identical and nearly identical nucleotide sequences were found between strains isolated more than 10 (GA5) and 25 (GA2) years apart. The A-terminal part of they G protein of genotype GA2 was highly conserved. In contrast, the A-terminal part of the G protein of genotype GA5 exhibited a pronounced variation in its amino acid sequence over time.

Six different genotypes of mumps virus, A, C, D, G, H and I, genotyped on the basis of the small hydrophobic protein gene sequence, were subjected to antigenic comparison. Monoclonal antibodies directed against the haemagglutinin-neuraminidase protein of the SBL-1 strain of genotype A were used in immunofluorescence tests with different mumps virus strains. In addition, the six virus genotypes were compared by cross-neutralization tests with human post-vaccination sera after vaccination with the Jeryl Lynn (JL) strain of mumps virus and with rabbit hyperimmune sera directed against the A or D genotypes of mumps virus. Genotypes C, D, G, H and I could not be antigenically separated. In contrast, three different virus strains of genotype A, SBL-1, JL and Kilham, were distinct and were found to represent three different serotypes within the A genotype of mumps virus. Vaccination of Swedish children with the JL strain of mumps virus resulted in clearly lower neutralization titres against the SBL-1 strain, which is endemic in Sweden, compared to the homologous vaccine titres.

The nucleotide sequences of a 300 bp segment carrying the small hydrophobic (SH) protein gene of a large number of virus strains, belonging to 10 different mumps virus genotypes denoted A-J, were compared. When virus strains belonging to the same genotype were compared intra-genotypically, a variation in the range 2-4% was recorded. The level of inter-genotypical variation was higher (8-18%). A consistent finding was the more pronounced variation found when the A genotype was compared to the B-J genotypes than when the B-J genotypes were compared among themselves. A similar relatively more pronounced genetic variation between the A and B-D genotypes has also been found previously with the hemagglutinin-neuraminidase and fusion protein genes. It is concluded that the establishment of a new genotype should be founded on a nucleotide variation of the SH gene of > or = 6% in relation to previously described genotypes.

Twenty-nine Danish virus isolates and 14 serum samples from patients with mumps were genotyped by nucleotide sequencing of the small hydrophobic (SH) protein gene and the deduced 57 amino acid sequences were aligned with sequences of mumps virus strains published previously. Four neurovirulent genotypes of the SH protein gene, genotypes C, D, H and a new genotype, designated J, were found. There was a dynamic fluctuation of the different genotypes over the two decade period of time. Genotype J was found from 1981 to 1988; genotypes C and H exhibited a similar distribution in time. Genotype D was found between 1979 and 1982, it then disappeared and reappeared again in 1996. From 1996 onwards, genotype D was found to be the predominant genotype, which is in contrast to the situation seen in the neighbouring country of Sweden, where, since 1985, only genotype A has been found.

In April 1999, a previously healthy 22-year-old woman was taken ill with fever and bilateral swelling of the parotid glands. A chronic course of disease extending from April to December was found with swelling of the parotid glands, fatigue, low grade fever, episodes of tachycardia and nightswetting. Mumps virus RNA of genotype A character based on the SH (small hydrophobic) protein gene classification was demonstrated in three serum samples collected during the course of clinical disease. Different criteria for reinfection were fulfilled including demonstration of IgG antibodies by ELISA in a preinfection serum sample. The preinfection serum sample of the patient was able to efficiently neutralize the infectivity of a heterologous genotype D strain but was unable to neutralize the homologous genotype A virus. The findings in the present study may offer an explanation of a mechanism behind previously observed vaccine failures and the occurrence of reinfection with heterologous mumps virus strains.

One of the most contentious topics in the study of human evolution is that of the time, place and mode of origin of Homo sapiens. The discovery in the Northern Danakil (Afar) Depression, Eritrea, of a well-preserved Homo cranium with a mixture of characters typical of H. erectus and H. sapiens contributes significantly to this debate. The cranium was found in a succession of fluvio-deltaic and lacustrine deposits and is associated with a rich mammalian fauna of early to early-middle Pleistocene age. A magnetostratigraphic survey indicates two reversed and two normal magnetozones. The layer in which the cranium was found is near the top of the lower normal magnetozone, which is identified as the Jaramillo subchron. Consequently, the human remains can be dated at approximately 1 million years before present.