Plasma lipidome reveals critical illness and recovery from human Ebola virus disease.

Ebola virus disease (EVD) often leads to severe and fatal outcomes in humans with early supportive care increasing the chances of survival. Profiling the human plasma lipidome provides insight into critical illness as well as diseased states, as lipids have essential roles as membrane structural components, signaling molecules, and energy sources. Here we show that the plasma lipidomes of EVD survivors and fatalities from Sierra Leone, infected during the 2014-2016 Ebola virus outbreak, were profoundly altered. Focusing on how lipids are associated in human plasma, while factoring in the state of critical illness, we found that lipidome changes were related to EVD outcome and could identify states of disease and recovery. Specific changes in the lipidome suggested contributions from extracellular vesicles, viremia, liver dysfunction, apoptosis, autophagy, and general critical illness, and we identified possible targets for therapies enhancing EVD survival.

Medication-related osteonecrosis of the jaw after tooth extraction in cancer patients: a multicenter retrospective study.

Root amputation, immunosuppressive therapy, mandibular tooth extraction, pre-existing inflammation, and longer duration of treatment with bone-modifying agents were significantly associated with an increased risk of medication-related osteonecrosis of the jaw. Hopeless teeth should be extracted without drug holiday before the development of inflammation in cancer patients receiving high-dose bone-modifying agents. INTRODUCTION: No studies have comprehensively analyzed the influence of pre-existing inflammation, surgical procedure-related factors such as primary wound closure, demographic factors, and drug holiday on the incidence of medication-related osteonecrosis of the jaw (MRONJ). The purpose of this study was to retrospectively investigate the relationships between these various factors and the development of MRONJ after tooth extraction in cancer patients receiving high-dose bone-modifying agents (BMAs) such as bisphosphonates or denosumab. METHODS: Risk factors for MRONJ after tooth extraction were evaluated with univariate and multivariate analyses. The following parameters were investigated in all patients: demographics, type and duration of BMA use, whether BMA use was discontinued before tooth extraction (drug holiday), the duration of such discontinuation, the presence of pre-existing inflammation, and whether additional surgical procedures (e.g., incision, removal of bone edges, root amputation) were performed. RESULTS: We found that root amputation (OR = 22.62), immunosuppressive therapy (OR = 16.61), extraction of mandibular teeth (OR = 12.14), extraction of teeth with pre-existing inflammation, and longer duration (≥ 8 months) of high-dose BMA (OR = 7.85) were all significantly associated with MRONJ. CONCLUSIONS: Tooth extraction should not necessarily be postponed in cancer patients receiving high-dose BMA. The effectiveness of a short-term drug holiday was not confirmed, as drug holidays had no significant impact on MRONJ incidence. Tooth extraction may be acceptable during high-dose BMA therapy until 8 months after initiation.

Avian influenza virus transmission to mammals.

Influenza A viruses cause yearly epidemics and occasional pandemics. In addition, zoonotic influenza A viruses sporadically infect humans and may cause severe respiratory disease and fatalities. Fortunately, most of these viruses do not have the ability to be efficiently spread among humans via aerosols or respiratory droplets (airborne transmission) and to subsequently cause a pandemic. However, adaptation of these zoonotic viruses to humans by mutation or reassortment with human influenza A viruses may result in airborne transmissible viruses with pandemic potential. Although our knowledge of factors that affect mammalian adaptation and transmissibility of influenza viruses is still limited, we are beginning to understand some of the biological traits that drive airborne transmission of influenza viruses among mammals. Increased understanding of the determinants and mechanisms of airborne transmission may aid in assessing the risks posed by avian influenza viruses to human health, and preparedness for such risks. This chapter summarizes recent discoveries on the genetic and phenotypic traits required for avian influenza viruses to become airborne transmissible between mammals.

Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013.

Novel influenza viruses of the H7N9 subtype have infected 33 and killed nine people in China as of 10 April 2013. Their haemagglutinin (HA) and neuraminidase genes probably originated from Eurasian avian influenza viruses; the remaining genes are closely related to avian H9N2 influenza viruses. Several characteristic amino acid changes in HA and the PB2 RNA polymerase subunit probably facilitate binding to human-type receptors and efficient replication in mammals, respectively, highlighting the pandemic potential of the novel viruses.

Emerging and reemerging influenza virus infections.

Influenza A virus infection occurs in many species. Wild waterfowl harbor the widest variety of influenza A viruses and serve as a constant reservoir for the emergence of new viruses. Highly pathogenic avian influenza, or "fowl plague," has been a known poultry disease for more than 130 years. It continues to emerge and reemerge, but global changes in trade and poultry production have expanded the impact and geographic range of these outbreaks. One subtype of highly pathogenic avian influenza, H5N1, has infected poultry on several continents as well as many people, leading to a human disease that is markedly different from seasonal influenza and that is associated with high mortality.

Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses.

In 1997, an H5N1 influenza A virus was transmitted from birds to humans in Hong Kong, killing 6 of the 18 people infected. When mice were infected with the human isolates, two virulence groups became apparent. Using reverse genetics, we showed that a mutation at position 627 in the PB2 protein influenced the outcome of infection in mice. Moreover, high cleavability of the hemagglutinin glycoprotein was an essential requirement for lethal infection.

Risk assessment of recent Egyptian H5N1 influenza viruses.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are enzootic in poultry populations in different parts of the world, and have caused numerous human infections in recent years, particularly in Egypt. However, no sustained human-to-human transmission of these viruses has yet been reported. We tested nine naturally occurring Egyptian H5N1 viruses (isolated in 2014-2015) in ferrets and found that three of them transmitted via respiratory droplets, causing a fatal infection in one of the exposed animals. All isolates were sensitive to neuraminidase inhibitors. However, these viruses were not transmitted via respiratory droplets in three additional transmission experiments in ferrets. Currently, we do not know if the efficiency of transmission is very low or if subtle differences in experimental parameters contributed to these inconsistent results. Nonetheless, our findings heighten concern regarding the pandemic potential of recent Egyptian H5N1 influenza viruses.

The pathogenesis of Ebola hemorrhagic fever.

Ebola virus causes lethal hemorrhagic disease in humans, yet there are still no satisfactory biological explanations to account for its extreme virulence. This review focuses on recent findings relevant to understanding the pathogenesis of Ebola virus infection and developing vaccines and effective therapy. The available data suggest that the envelope glycoprotein and the interaction of some viral proteins with the immune system are likely to play important roles in the extraordinary pathogenicity of this virus. There are also indications that genetically engineered vaccines, including plasmid DNA and viral vectors expressing Ebola virus proteins, and passive transfer of neutralizing antibodies could be feasible options for the control of Ebola virus-associated disease.

Reverse genetics systems for the generation of segmented negative-sense RNA viruses entirely from cloned cDNA.

Reverse genetics is defined as the generation of virus entirely from cloned cDNA. For negative-sense RNA viruses, whose genomes are complementary to mRNA in their orientation, the viral RNA(s) and the viral proteins required for replication and translation must be provided to initiate the viral replication cycle. Segmented negative-sense RNA viruses were refractory to genetic manipulation until 1989. In this chapter, we review developments in the reverse genetics of segmented negative-sense RNA viruses, beginning with the in vitro reconstitution of viral polymerase complexes in the late 1980s and culminating in the generation of Bunyamwera and influenza virus entirely from plasmid DNA almost a decade later.

Orthomyxovirus replication, transcription, and polyadenylation.

Efficient in vitro and in vivo systems are now in place to study the role of viral proteins in replication and/or transcription, the regulation of these processes, polyadenylation of viral mRNAs, the viral promoter structures, or the significance of noncoding regions for virus replication. In this chapter, we review the status of current knowledge of the orthomyxovirus RNA synthesis.

Swine influenza virus strains recognize sialylsugar chains containing the molecular species of sialic acid predominantly present in the swine tracheal epithelium.

We determined the ratio of N-glycolylneuraminic acid (Neu5Gc) to N-acetylneuraminic acid (Neu5Ac) in swine respiratory epithelia by fluorometric high-performance liquid chromatography, and examined the binding specificity of swine influenza virus strains for gangliosides containing different molecular species of sialic acid (Neu5Ac and Neu5Gc), and for bovine erythrocyte sialoglycoprotein 2 (GP-2) containing Neu5Gc as its predominate sialic acid (96% of total sialic acids). The presence of Neu5Gc, which had not been detected in human tracheal epithelia, and Neu5Ac in swine tracheal epithelia was observed in a 1:1 ratio. The swine influenza virus H1 and H3 isolates tested, except for A/swine/Iowa/15/30 (H1N1), displayed a marked binding ability for sialylsugar chains containing Neu5Gc compared with that of the human influenza virus strains. These results suggest that swine influenza viruses recognize sialylsugar chains containing the molecular species of sialic acid present predominantly in the swine tracheal epithelium.

Substitution of amino acid residue in influenza A virus hemagglutinin affects recognition of sialyl-oligosaccharides containing N-glycolylneuraminic acid.

Sialic acids are essential components of cell surface receptors used by influenza viruses. To determine the molecular mechanisms of viral recognition of two major species of sialic acids, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), we tested the binding reactivity of nine human H3 influenza A viruses to sialylglycolipids containing type II sugar chain and different molecular species of terminal sialic acids. All human H3 viruses tested except A/Memphis/1/71 bound both Neu5Ac and Neu5Gc. Nucleotide sequence analysis suggests that amino acids at 143, 155, and 158 are linked to the viral recognition of Neu5Gc.

Biologic effects of introducing additional basic amino acid residues into the hemagglutinin cleavage site of a virulent avian influenza virus.

We mutated the virulent avian influenza virus A/turkey/Ontario/7732/66 (H5N9)[Q-R-R-R-K-K-R\G at the hemagglutinin (HA) cleavage site] to create a mutant, R(MO-0), with additional basic residues at this site (Q-R-R-R-R-R-K-K-R\G) by reverse genetics. When tested in chicken embryo fibroblast culture, this mutant showed reduced HA cleavability compared to that of the wild-type virus, but its plaque size was not appreciably altered. Virulence of the R(MO-0) virus in chickens was lower than that of the wild-type virus. These findings indicate that addition of excessive basic residues to an optimal recognition sequence for HA cleavage enzymes at the cleavage site is deleterious for HA cleavability. Previously, we showed that a mutant containing the suboptimal HA cleavage site sequence for cleavage enzyme recognition also had reduced HA cleavability and virulence compared to the wild-type virus. We conclude that the data presented here further substantiate our belief that the level of HA cleavability correlates with the degree of virulence when all other genetic characteristics are considered equal, irrespective of the mechanisms by which HA cleavability is reduced.

Nucleotide sequence of the avian influenza A/Mallard/NY/6750/78 virus polymerase genes.

The avian influenza A/Mallard/NY/6750/78 virus is currently being evaluated as a donor of attenuating genes in the construction of live avian-human influenza A reassortant virus vaccines for use in humans. We determined the nucleotide sequences of the three polymerase gene segments of this virus. This completes the nucleotide sequence of the six transferrable genes of the avian donor virus. Comparison of the nucleotide and deduced amino acid sequences of the non-glycoprotein genes of the avian A/Mallard/78 virus with representative avian and human influenza A viruses suggests that the PB1 gene of H2N2 subtype human influenza A viruses may have been derived from a non-human, possibly avian influenza A virus by genetic reassortment. In addition, several regions of conserved amino acids with potential functional significance were identified in the deduced amino acid sequences of the polymerase proteins.

Are there alternative avian influenza viruses for generation of stable attenuated avian-human influenza A reassortant viruses?

The present study evaluated gull influenza A viruses as donors of attenuating genes for the production of live, attenuated influenza A H1N1 and H3N2 avian-human (ah) reassortant viruses for use as vaccines to prevent disease due to influenza A viruses in humans. The previously evaluated duck influenza A viruses were abandoned as donors of attenuating avian influenza virus genes because clinical evaluation of H1N1 and H3N2 ah reassortant virus vaccines derived from duck viruses documented residual virulence of H1N1 reassortants for seronegative infants and young children. Gull influenza A viruses occupy an independent ecologic niche and are rarely isolated from species other than gulls. The possibility of using gull influenza A viruses as donors of internal gene segments in ah reassortant viruses was evaluated in the present study using three different gull viruses and three human influenza A viruses. Gull-human H3N2 reassortant influenza A viruses with the desired 6-2 genotype (six internal avian influenza virus genes and the two human influenza virus surface glycoprotein genes) were readily generated and were found to be attenuated for squirrel monkeys and chimpanzees. However, ah reassortant viruses with gull and human influenza A H1N1 genes were difficult to generate, and reassortants that had the desired genotype of six gull virus genes with human influenza A H1 and N1 genes were not isolated despite repeated attempts. The gull PB2, NP and NS genes were not present in any of the gull-human H1N1 reassortants generated. The under-representation of these three gene segments suggests that reassortants bearing one or more of these three gene segments might have reduced viability indicative of a functional incompatibility in their gene products. The difficulties encountered in the generation of a 6-2 gull-human H1N1 reassortant virus are sufficient to conclude that the gull influenza A viruses tested would not be useful as donors of sets of six internal genes to attenuate human influenza A viruses. This study also identifies influenza virus gene segments that appear to be incompatible for generation of reassortants. Elucidation of the molecular basis of this restriction may provide information on intergenic interactions involved in virion assembly or packaging.

The HA1 of cold-adapted influenza B vaccine is not altered during replication in human vaccinees.

Influenza viruses recovered from 14 children 2-10 days after vaccination with an egg-grown, cold-adapted influenza B vaccine (B/AA/1/86) were analyzed. Hemagglutination-inhibition (HI) assays using monoclonal antibodies did not detect antigenic differences between the vaccine strain and the viruses recovered from the vaccinees. Furthermore, nucleotide sequence analysis of the HA1 region did not reveal any changes compared to the sequence of the vaccine strain. These findings indicate that influenza B vaccine hemagglutinin is genetically stable during replication in human vaccinees.

Changes in H3 influenza A virus receptor specificity during replication in humans.

Influenza A viruses of the H3 subtype caused the 1968 Hong Kong pandemic, the hemagglutinin (HA) gene being introduced into humans following a reassortment event with an avian virus. Receptor specificity and serum inhibitor sensitivity of the HA of influenza A viruses are linked to the host species. Human H3 viruses preferentially recognize N-acetyl sialic acid linked to galactose by alpha2,6 linkages (Neu5Acalpha2,6Gal) and are sensitive to serum inhibitors, whereas avian and equine viruses preferentially recognize Neu5Acalpha2,3Gal linkages and are resistant to serum inhibitors. We have examined the receptor specificity and serum inhibitor sensitivity of H3 human influenza A viruses from the time they were introduced into the human population to gain insight into the mechanism of viral molecular evolution and host tropism. All of the viruses were sensitive to neutralization and hemagglutination inhibition by horse serum. Early H3 viruses were resistant to pig and rabbit serum inhibitors. Viruses isolated after 1977 were uniformly sensitive to inhibition by pig and rabbit sera. The recognition of Neu5Acalpha2,3Gal or Neu5Acalpha2,6Gal linkages was not correlated with the serum sensitivity. These data showed that the receptor specificity of HA, measured as inhibitor sensitivity, has changed during replication in humans since its introduction from an avian virus.

Influence of host species on the evolution of the nonstructural (NS) gene of influenza A viruses.

The matrix (M) and nonstructural (NS) genes of influenza A viruses each encode two overlapping proteins. In the M gene, evolution of one protein affects that of the other. To determine whether or not this evolutionary influence operating between the two M proteins also occurs in the NS gene, we sequenced the NS genes of 36 influenza A viruses isolated from a broad spectrum of animal species (wild and domestic birds, horses, pigs, humans, and sea mammals) and analyzed them phylogenetically, together with other previously published sequences. These analyses enabled us to conclude the following host species-related points that are not found in the other influenza A virus genes and their gene products. (1) The evolution of the two overlapping proteins encoded by the NS gene are lineage-dependent, unlike the M gene where evolutionary constraints on the Ml protein affect the evolution of the M2 protein (Ito et al.. J. Virol. 65 (1991) 5491 5498). (2) The gull-specific lineage contained nonH13 gull viruses and the non-gull avian lineage contained H13 gull viruses, indicating that the gull-specific lineage does not link to the H13 HA subtype in the NS gene unlike findings with other genes. (3) The branching topology of the recent equine lineage (H7N7 viruses isolated after 1973 and H3N8) indicates recent introduction of the NS, M, and PB2 genes into horses from avian sources by genetic reassortment.

Isolation and use of eight phages for typing Yersinia enterocolitica O3.

Subdivision of 137 isolates of Yersinia enterocolitica O3 into eight phagovars has been achieved. Some geographical differences were found in the sources of these phagovars and also of two biovars.