Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1.

Influenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral nonstructural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host species barrier. We characterized the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionarily distinct NS1 proteins counteract the IFN response using different and mutually exclusive mechanisms: while the NS1 proteins of early EIVs block general gene expression by binding to cellular polyadenylation-specific factor 30 (CPSF30), NS1 proteins from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and were rapidly selected for during EIV evolution, highlighting the importance of evolutionary processes in immune evasion mechanisms during IAV adaptation.IMPORTANCE Influenza A viruses (IAVs) infect certain avian reservoir species and occasionally transfer to and cause epidemics of infections in some mammalian hosts. However, the processes by which IAVs gain the ability to efficiently infect and transmit in mammals remain unclear. H3N8 equine influenza virus (EIV) is an avian-origin virus that successfully established a new lineage in horses in the early 1960s and is currently circulating worldwide in the equine population. Here, we analyzed the molecular evolution of the virulence factor nonstructural protein 1 (NS1) and show that NS1 proteins from different time periods after EIV emergence counteract the host innate immune response using contrasting strategies, which are associated with two mutations that appeared sequentially during EIV evolution. The results shown here indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.

Genetic Analysis of Feline Panleukopenia Virus Full-length VP2 Gene in Domestic Cats Between 2006-2008 and 2012-2014, Portugal.

Feline panleukopenia virus (FPV) and canine parvovirus (CPV) are two closely related viruses, which cause acute gastroenteritis in carnivores, and cats may be infected by strains of both viruses. The viruses are found worldwide and may have changing host ranges and genetic variation that can be found around the world in some cases. Here, we screened a Portuguese population of cats by a conventional PCR assay for the presence of FPV/CPV viruses in faecal samples and tissues between 2006-2008 and 2012-2014. The sequence analysis of the complete VP2 gene showed that 18 of 31 animals tested were positive for FPV DNA, and no case of CPV infection was detected. The analysis of specific DNA detected three new non-synonymous substitutions in the VP2 gene that were found in single groups and were related to viruses reported elsewhere by phylogenetic analysis - some were related to Italian isolates, one was closely related to isolates from Vietnam and China, and two were related with older strains from the USA. The results of our study show that FPV circulated in the Portuguese cat population and as expected the found strains are slightly divergent from those reported previously.

Anticipating the species jump: surveillance for emerging viral threats.

Zoonotic disease surveillance is typically triggered after animal pathogens have already infected humans. Are there ways to identify high-risk viruses before they emerge in humans? If so, then how and where can identifications be made and by what methods? These were the fundamental questions driving a workshop to examine the future of predictive surveillance for viruses that might jump from animals to infect humans. Virologists, ecologists and computational biologists from academia, federal government and non-governmental organizations discussed opportunities as well as obstacles to the prediction of species jumps using genetic and ecological data from viruses and their hosts, vectors and reservoirs. This workshop marked an important first step towards envisioning both scientific and organizational frameworks for this future capability. Canine parvoviruses as well as seasonal H3N2 and pandemic H1N1 influenza viruses are discussed as exemplars that suggest what to look for in anticipating species jumps. To answer the question of where to look, prospects for discovering emerging viruses among wildlife, bats, rodents, arthropod vectors and occupationally exposed humans are discussed. Finally, opportunities and obstacles are identified and accompanied by suggestions for how to look for species jumps. Taken together, these findings constitute the beginnings of a conceptual framework for achieving a virus surveillance capability that could predict future species jumps.

Gastroparesis and functional dyspepsia: excerpts from the AGA/ANMS meeting.

BACKGROUND: Despite the relatively high prevalence of gastroparesis and functional dyspepsia, the aetiology and pathophysiology of these disorders remain incompletely understood. Similarly, the diagnostic and treatment options for these two disorders are relatively limited despite recent advances in our understanding of both disorders. PURPOSE: This manuscript reviews the advances in the understanding of the epidemiology, pathophysiology, diagnosis, and treatment of gastroparesis and functional dyspepsia as discussed at a recent conference sponsored by the American Gastroenterological Association (AGA) and the American Neurogastroenterology and Motility Society (ANMS). Particular focus is placed on discussing unmet needs and areas for future research.

Treatment of gastroparesis: a multidisciplinary clinical review.

This clinical review on the treatment of patients with gastroparesis is a consensus document developed by the American Motility Society Task Force on Gastroparesis. It is a multidisciplinary effort with input from gastroenterologists and other specialists who are involved in the care of patients with gastroparesis. To provide practical guidelines for treatment, this document covers results of published research studies in the literature and areas developed by consensus agreement where clinical research trials remain lacking in the field of gastroparesis.

Flow cytometric characterization of perfused human bone marrow cultures: identification of the major cell lineages and correlation with the CFU-GM assay.

BACKGROUND: Prolific cultures of human bone marrow mononuclear cells (BM MNCs) were recently developed that include a full spectrum of hematopoietic and accessory cells, with the presence of autofluorescent cells indicating adequate cell expansion. However, phenotypic and functional clonogenic characterizations of the autofluorescent cells and the various other subpopulations present in these cultures have not been carried out. METHODS: Cells from a continuously perfused bioreactor inoculated with BM MNCs and cultured for 12 days in serum-containing medium with PIXY321, erythropoietin, and with or without FLT3-L were evaluated by using flow cytometry. RESULTS: Two antibodies, CD71 and CD13, allowed the separation of the autofluorescent cells into two distinct populations. The CD71+CD13++ autofluorescent population contained the colony-forming unit (CFU) fibroblast, and the CD71++CD13++ autofluorescent population contained macrophage/dendritic like cells. The CFU-granulocyte/macrophage (CFU-GM) could not be thoroughly evaluated with CD71 and CD13. However, the number of CD13+/++Lin- cells correlated with the number of CFU-GM (r = 0.83), with approximately 1 CFU-GM for every 30 CD13+/++Lin- cells. CONCLUSIONS: The data showed that CD71 and CD13 antibodies separate the autofluorescent cells into two populations but do not separate hematopoietic cells into specific phenotypic populations. The data also showed that the number of CD13+/++Lin- cells correlated with the number of CFU-GM. These data present the initial step toward detailed phenotypic analysis of ex vivo expanded human BM MNC cultures.

Parvovirus infections in wild carnivores.

Various parvoviruses infect carnivores and can cause disease. In this review article the knowledge about infections of free-ranging or captive carnivores with the feline parvoviruses, feline panleukopenia virus, and canine parvovirus, including the antigenic types CPV-2a and -2b, as well as Aleutian disease of mink virus and minute virus of canines are summarized. Particular emphasis is placed on description of the evolution of canine parvovirus which apparently involved wild carnivore hosts.

Canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells.

Canine parvovirus (CPV) enters and infects cells by a dynamin-dependent, clathrin-mediated endocytic pathway, and viral capsids colocalize with transferrin in perinuclear vesicles of cells shortly after entry (J. S. L. Parker and C. R. Parrish, J. Virol. 74:1919-1930, 2000). Here we report that CPV and feline panleukopenia virus (FPV), a closely related parvovirus, bind to the human and feline transferrin receptors (TfRs) and use these receptors to enter and infect cells. Capsids did not detectably bind or enter quail QT35 cells or a Chinese hamster ovary (CHO) cell-derived cell line that lacks any TfR (TRVb cells). However, capsids bound and were endocytosed into QT35 cells and CHO-derived TRVb-1 cells that expressed the human TfR. TRVb-1 cells or TRVb cells transiently expressing the feline TfR were susceptible to infection by CPV and FPV, but the parental TRVb cells were not. We screened a panel of feline-mouse hybrid cells for susceptibility to FPV infection and found that only those cells that possessed feline chromosome C2 were susceptible. The feline TfR gene (TRFC) also mapped to feline chromosome C2. These data indicate that cell susceptibility for these viruses is determined by the TfR.

Canine parvovirus capsid assembly and differences in mammalian and insect cells.

We examined the assembly processes of the capsid proteins of canine parvovirus (CPV) in mammalian and insect cells. In CPV-infected cells empty capsids assembled within 15 min, and then continued to form over the following 1 h, while full (DNA-containing) capsids were detected only after 60 min, and those accumulated slowly over several hours. In cells expressing VP1 and VP2 or only VP2, empty capsid formation was also efficient, but was slightly slower than that in infected cells. Small amounts of trimer forms of VP2 were detected in cells expressing wild type capsid proteins, but were not seen for mutants containing changes that prevented capsid assembly. CPV capsids accumulated in the cell nucleus, but mutant VP1 and VP2 proteins that did not assemble became distributed throughout the nucleus and the cytoplasm, irrespective of whether they were expressed as VP1 and VP2, or as VP2 only. Urea or pH treatment of empty capsids released dimer, trimer, or pentamer capsid protein combinations, while treatment of full capsids consistently released trimer and, in some cases, pentamer forms. When wild type or assembly-defective VP2 genes were expressed from recombinant baculoviruses in insect cells, most of the protein was recovered as noncapsid aggregates, and only a small proportion assembled into capsids. Both the assembled capsids and the noncapsid aggregates were seen primarily in the cytoplasm of the insect cells. The VP2 expressed in insect cells that was recovered in aggregates had an isoelectric point of about pH 6.3, while that recovered from assembled capsids had a pI of about 5.2, similar to that seen for the VP2 of capsids recovered from mammalian cells.

Host range and variability of calcium binding by surface loops in the capsids of canine and feline parvoviruses.

Canine parvovirus (CPV) emerged in 1978 as a host range variant of feline panleukopenia virus (FPV). This change of host was mediated by the mutation of five residues on the surface of the capsid. CPV and FPV enter cells by endocytosis and can be taken up by many non-permissive cell lines, showing that their host range and tissue specificity are largely determined by events occurring after cell entry. We have determined the structures of a variety of strains of CPV and FPV at various pH values and in the presence or absence of Ca(2+). The largest structural difference was found to occur in a flexible surface loop, consisting of residues 359 to 375 of the capsid protein. This loop binds a divalent calcium ion in FPV and is adjacent to a double Ca(2+)-binding site, both in CPV and FPV. Residues within the loop and those associated with the double Ca(2+)-binding site were found to be essential for virus infectivity. The residues involved in the double Ca(2+)-binding site are conserved only in FPV and CPV. Our results show that the loop conformation and the associated Ca(2+)-binding are influenced by the Ca(2+) concentration, as well as pH. These changes are correlated with the ability of the virus to hemagglutinate erythrocytes. The co-localization of hemagglutinating activity and host range determinants on the virus surface implies that these properties may be functionally linked. We speculate that the flexible loop and surrounding regions are involved in binding an as yet unidentified host molecule and that this interaction influences host range.

Cytoplasmic trafficking of the canine parvovirus capsid and its role in infection and nuclear transport.

To begin a successful infection, viruses must first cross the host cell plasma membrane, either by direct fusion with the membrane or by receptor-mediated endocytosis. After release into the cytoplasm those viruses that replicate in the nucleus must target their genome to that location. We examined the role of cytoplasmic transport of the canine parvovirus (CPV) capsid in productive infection by microinjecting two antibodies that recognize the intact CPV capsid into the cytoplasm of cells and also by using intracellular expression of variable domains of a neutralizing antibody fused to green fluorescence protein. The two antibodies tested and the expressed scFv all efficiently blocked virus infection, probably by binding to virus particles while they were in the cytoplasm and before entering the nucleus. The injected antibodies were able to block most infections even when injected 8 h after virus inoculation. In control studies, microinjected capsid antibodies did not interfere with CPV replication when they were coinjected with an infectious plasmid clone of CPV. Cytoplasmically injected full and empty capsids were able to move through the cytosol towards the nuclear membrane in a process that could be blocked by nocodazole treatment of the cells. Nuclear transport of the capsids was slow, with significant amounts being found in the nucleus only 3 to 6 h after injection.

Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking.

Canine parvovirus (CPV) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. We used a variety of approaches to define the early stages of cell entry by CPV. Electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. Overexpression of a dominant interfering mutant of dynamin in the cells altered the trafficking of capsid-containing vesicles. There was a 40% decrease in the number of CPV-infected cells in mutant dynamin-expressing cells, as well as a approximately 40% decrease in the number of cells in S phase of the cell cycle, which is required for virus replication. However, there was also up to 10-fold more binding of CPV to the surface of mutant dynamin-expressing cells than there was to uninduced cells, suggesting an increased receptor retention on the cell surface. In contrast, there was little difference in virus binding, virus infection rate, or cell cycle distribution between induced and uninduced cells expressing wild-type dynamin. CPV particles colocalized with transferrin in perinuclear endosomes but not with fluorescein isothiocyanate-dextran, a marker for fluid-phase endocytosis. Cells treated with nanomolar concentrations of bafilomycin A1 were largely resistant to infection when the drug was added either 30 min before or 90 min after inoculation, suggesting that there was a lag between virus entering the cell by clathrin-mediated endocytosis and escape of the virus from the endosome. High concentrations of CPV particles did not permeabilize canine A72 or mink lung cells to alpha-sarcin, but canine adenovirus type 1 particles permeabilized both cell lines. These data suggest that the CPV entry and infection pathway is complex and involves multiple vesicular components.

Early stages of influenza virus entry into Mv-1 lung cells: involvement of dynamin.

Viruses generally have one of two mechanisms for entry and uncoating. They can enter the cell either by endocytosis or by direct fusion at the plasma membrane. We have established a novel mink lung (Mv-1) cell line that expresses a dominant-interfering form of dynamin-1 (K44A) under the control of a tetracycline-responsive element and studied the early events in influenza infection using these cells. We found that influenza virus binds equally to both induced and uninduced cells, but in K44A-expressing cells, electron microscopy showed viruses trapped in deep coated pits and irregular-shaped tubular structures that contain discrete coated regions. We also show by immunofluorescence and confocal microscopy that entry of incoming virus into the nucleus is blocked in K44A-expressing cells. Virus replication was assayed by immunofluorescence microscopy and was strongly inhibited at both early and late times postinfection in K44A-expressing cells. Virus infectivity was inhibited by approximately 2 log units in cells expressing K44A dynamin when analyzed by influenza plaque assay. Overall these data show that dynamin is required for efficient influenza virus entry, presumably due to its function in release of vesicles from coated pits.

Comparison of two single-chain antibodies that neutralize canine parvovirus: analysis of an antibody-combining site and mechanisms of neutralization.

We cloned the heavy- and light-chain variable domains of two monoclonal antibodies that recognize each of the two major neutralizing antigenic sites of the canine parvovirus (CPV) capsid. After expression in Escherichia coli as single-chain variable domains (scFv) with glycine-serine linker sequences, both scFv bound CPV capsids with the same specificity as the intact IgG, but with 10- to 20-fold lower avidity. Both scFvs neutralized CPV infectivity with efficiency similar to that of the IgG. Although both IgGs inhibited hemagglutination by CPV, only one scFv was inhibiting. The binding of one of the antibodies has previously been analyzed by cryoelectron microscopic reconstruction and the epitope-binding residues predicted. Mutagenesis of predicted contact residues in three heavy-chain complementarity-determining regions (CDR) showed that mutants of CDR1 or CDR3 reduced the binding of the scFv by about 10-fold compared with the wild-type scFv, while no effect was seen for one mutant of CDR2. The levels of neutralization of CPV and of hemagglutination inhibition by the scFv mutants were proportional to their reduction in binding affinity compared with the wild type. Neither scFv blocked virus binding to host cells, but they both caused aggregation of the capsids and appeared to affect the process of infection after virus uptake into the cells.

Host range relationships and the evolution of canine parvovirus.

Canine parvovirus (CPV) is an example of an unusual class of emerging virus-those that gain an altered host range through genetic variation and subsequently become widespread pathogens of their new and previously resistant host species. CPV was first detected in 1978 as the cause of new diseases in dogs throughout the world, when it rapidly spread throughout domestic populations, as well as becoming widespread in wild dogs. CPV was soon shown to be a variant of the long recognized feline panleukopenia virus (FPV), from which it differed in less than 1% at the nucleotide sequence level. Genetic analysis showed that virtually all of the biological differences between CPV and FPV, including the canine host range, were determined by three or four sequence differences in the viral capsid protein gene. Analysis of the atomic structures of the CPV and FPV capsids showed that the differences controlling host range were located within two different structural regions and were exposed on the capsid surface. The CPV which first emerged in 1978 appeared to be derived from a single ancestral sequence, which has allowed the ready analysis of the subsequent evolution of the virus in nature. Sequence analysis has also revealed that CPV strains have undergone a series of evolutionary selections in nature which have resulted in the global distribution of new virus variants. This was first seen in the global replacement between 1979 and 1981 of the original (1978) strain of the virus by a genetically and antigenically variant strain, and the subsequent widespread selection of other variants which have also become globally distributed. The genetic and antigenic variation in the virus strains was also correlated with changes in the host range of the virus, in particular in the ability to replicate in cats, and in canine host range differences seen in tissue culture cells.

Feline calicivirus capsid protein expression and self-assembly in cultured feline cells.

Feline calicivirus (FCV) capsid protein was expressed in feline cells employing the vaccinia virus MVA/T7 RNA polymerase system. The precursor protein was processed to a mature size protein that assembled to virus like particles (VLPs).

A heterogeneous nuclear ribonucleoprotein A/B-related protein binds to single-stranded DNA near the 5' end or within the genome of feline parvovirus and can modify virus replication.

Phage display of cDNA clones prepared from feline cells was used to identify host cell proteins that bound to DNA-containing feline panleukopenia virus (FPV) capsids but not to empty capsids. One gene found in several clones encoded a heterogeneous nuclear ribonucleoprotein (hnRNP)-related protein (DBP40) that was very similar in sequence to the A/B-type hnRNP proteins. DBP40 bound specifically to oligonucleotides representing a sequence near the 5' end of the genome which is exposed on the outside of the full capsid but did not bind most other terminal sequences. Adding purified DBP40 to an in vitro fill-in reaction using viral DNA as a template inhibited the production of the second strand after nucleotide (nt) 289 but prior to nt 469. DBP40 bound to various regions of the viral genome, including a region between nt 295 and 330 of the viral genome which has been associated with transcriptional attenuation of the parvovirus minute virus of mice, which is mediated by a stem-loop structure of the DNA and cellular proteins. Overexpression of the protein in feline cells from a plasmid vector made them largely resistant to FPV infection. Mutagenesis of the protein binding site within the 5' end viral genome did not affect replication of the virus.

Nutrition support for the mechanically ventilated patient.

Nutrition support is a hotly debated topic in most intensive care units. Is enteral nutrition or TPN best? Is gastric or small-bowel feeding safer? Are specialized formulas needed? These are only some of the issues, and the fact remains that there is a paucity of clear, solid data. Folklore has become the standard of practice in many areas of medicine; it is richly found in nutrition support. We must be careful not to get caught up in the trappings of our beliefs about nutrition support. Instead, we must continue to evaluate our own practices and fine-tune our skills of clinical assessment and common sense.