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.

Natural variation of canine parvovirus.

Canine parvovirus was first recognized during 1978. Analysis of isolates collected since its emergence revealed that viruses circulating after 1980 were antigenically different from earlier isolates. Monoclonal antibodies clearly distinguished the two strains, some being specific for either the old or the new viruses. Restriction enzyme analysis of viral DNA's showed that the post-1980 viruses were similar to earlier isolates, but some restriction site differences were present in the new strain. These results suggest that the canine parvoviruses infecting dogs in the seven areas of the United States that were sampled derive from a variant virus that replaced the original strain during 1980.

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.

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.

The structure of a neutralized virus: canine parvovirus complexed with neutralizing antibody fragment.

BACKGROUND: Members of the Parvovirus genus cause a variety of diseases in mammals, including humans. One of the major defences against viral infection is the presence of neutralizing antibodies that prevent virus particles from infecting target cells. The mechanism of neutralization is not well understood. We therefore studied the structure of canine parvovirus (CPV) complexed with the Fab fragment of a neutralizing antibody, A3B10, using image reconstruction of electron micrographs of vitrified samples, together with the already known structure of CPV from X-ray crystallographic data. RESULTS: The structure of the complex of CPV with Fab A3B10 has been determined to 23 A resolution. The known CPV atomic structure was subtracted from the electron density of the complex, and the difference map was used to fit the atomic coordinates of a known Fab fragment, HyHEL-5. The long axis of each Fab molecule is oriented in a near radial direction, inclined away from the two-fold axes. The viral epitope consists of 14 amino acid residues found in loops 1, 2 and 3 on the capsid surface, which include previously identified escape mutations. CONCLUSIONS: The mode of Fab binding suggests that the A3B10 neutralizing antibody cannot bind bivalently to the capsid across the two-fold axes, consistent with the observation that whole A3B10 antibody readily precipitates CPV. Since Fab A3B10 can also neutralize the virus, mechanisms of neutralization such as interference with cell attachment, cell entry, or uncoating, must be operative.

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.

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.

Canine parvovirus infections in a colony of dogs.

A serological study of canine parvovirus (CPV) infections in a colony of dogs was conducted over a period of 8 months. Twenty-two of 24 adults samples initially had significant antibody titres to CPV. Nine litters of puppies were bled at fortnightly intervals and the sera tested for antibodies to CPV. Twenty-nine of 35 naturally PV infections observed were subclinical. A puppy in one litter developed vomiting and diarrhoea during the same period as seroconversion to CPV. Four puppies from a further litter developed histologically confirmed myocarditis. Serological testing of this latter litter indicated that the virus infections occurred between 2 weeks prior to and 1 week post whelping, and that clinical disease developed 20-40 days after viral infection. The mean half life of the decline of passively derived immunity to CPV was 8.3 days.

There is nothing permanent except change. The emergence of new virus diseases.

The sudden appearance of apparently new viruses with pathogenic potential is of fundamental importance in medical microbiology and a constant threat to humans and animals. The emergence of a "new" pathogen is not an isolated event, as for instance the frequent appearance of new influenza virus strains demonstrates. Often the new virus strains co-circulate with the older strains in a susceptible population, but a replacement of the older strains has been also observed. In rare instances the new viruses can cause dramatic epidemics or pandemics, such as those observed with the human immunodeficiency virus, canine parvovirus, or most recently, with the agent of bovine spongiform encephalopathy in the United Kingdom. The mechanisms of the emergence are not always clearly understood, but an altered host range appears to be a common event. Whether a true change in host range occurs, or whether the virus adapted to the host and replicated more efficiently, is often unknown. This review tries to summarize the facts that are known about a wide variety of "new" viruses of mammals, such as the simian, human and feline lentiviruses, the feline coronaviruses, the feline parvoviruses, the carnivore morbilliviruses, the influenza A viruses, and the transmissible spongiform encephalopathies. A particular emphasis will be put on the genetic mechanisms that might have taken place and that might have been responsible for their sudden appearance.

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).

Characterization of canine parvovirus (CPV) interactions with 3201 T cells: involvement of GPI-anchored protein(s) in binding and infection.

Binding of canine parvovirus (CPV) to the susceptible feline T cell line 3201 was quantitated by fluorescence-activated cell sorter (FACS) analysis. CPV bound to the cells in a dose-dependent manner, while no binding to the non-permissive MSB-1 avian lymphoma cell line was detected. Binding could be competitively inhibited by addition of excess unlabeled empty capsids, or by pre-incubation of virus with a CPV-specific monoclonal antibody. To characterize the biochemical nature of this binding, live cells were treated with a variety of enzymes prior to use in the binding assay. Treatment with neuraminidase removed a significant proportion of the wild-type virus binding activity, while both proteinase K and phosphatidylinositol-specific phospholipase C (PI-PLC) prevented binding of a non-hemagglutinating (non-HA), non-sialic acid binding mutant to 3201 cells. This suggests that CPV binds to sialic acid expressed on host cells as well as erythrocyte membranes, and that it also binds a protein moiety which is glycosylphosphatidylinositol (GPI)-anchored. The role of these components in CPV infection was also examined by pretreating cells with neuraminidase or PI-PLC prior to inoculating them with either wild-type CPV or the non-hemagglutinating mutant. Neuraminidase treatment had no effect on the ability of CPV to infect the cells, while infectivity was severely compromised by pretreating the cells with either proteinase K or PI-PLC. GPI-anchored proteins on 3201 cells were further characterized by Triton X-114 extraction and reactivity to anti-CRD after PI-PLC treatment.

Determination of the detection limit of the polymerase chain reaction for chicken infectious anemia virus.

Chicken infectious anemia virus (CIAV) DNA in infected cell cultures and chicken tissues was detected using a polymerase chain reaction (PCR) assay. The complete CIAV genome of several strains was amplified in two segments with two sets of primer pairs. The DNA segments of four CIAV strains and full-length Cux-1 strain DNA were cloned. After amplification, 100 original genome equivalents were detected by Southern hybridization. The sensitivity of the assay was enhanced considerably by performing a reamplification with nested primers. This modification permitted the detection of one molecule of CIAV DNA. Some problems of the assay and its possible application are discussed.

Comparisons of feline panleukopenia virus, canine parvovirus, raccoon parvovirus, and mink enteritis virus and their pathogenicity for mink and ferrets.

Parvoviruses from mink (mink enteritis virus [MEV]), cats (feline panleukopenia virus [FPV]), raccoons (raccoon parvovirus [RPV]), and dogs (canine parvovirus [CPV]) were compared. Restriction enzyme analysis of the viral replicative-form DNA revealed no consistent differences between FPV and RPV isolates, but CPV and MEV isolates could be distinguished readily from other virus types. Feline panleukopenia virus, RPV, and MEV, but not CPV, replicated to high titers in mink. However, on the first passage, disease and microscopic lesions were observed only in mink inoculated with MEV. Feline panleukopenia virus and RPV isolates replicated in ferrets, but disease or microscopic lesions were not observed. Feline panleukopenia virus and RPV isolates could be passaged repeatedly in mink and ferrets. Virulence of FPV and RPV isolates was low compared with that of MEV, and only a single mink inoculated with FPV or with RPV developed clinical disease on the sixth passage of virus.

Characterization of antigenic variation among mink enteritis virus isolates.

Three antigenic forms of natural field isolates of mink enteritis virus were revealed with a panel of monoclonal antibodies generated against the closely studied feline panleukopenia virus and canine parvovirus-2 virus. Two types (types 2 and 3) were shown to be closely related by agar-gel precipitin tests and by restriction enzyme mapping. However, types 2 and 3 differed from the type 1 isolates in the same tests. In cross-protection studies, inactivated viral vaccines made from any one of the 3 variant types of mink enteritis virus protected mink against challenge exposure by the homologous, as well as the heterologous, antigenic types.

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.

Antigenic and genetic analysis of canine parvoviruses in southern Africa.

Canine parvovirus (CPV) is a significant pathogen of domestic and free-ranging carnivores all over the world. It suddenly appeared at the end of the 1970s and most likely emerged as a variant of the well known feline panleukopenia virus (FPV). During its adaptation to the new host, the domestic-dog, the virus has changed its antigenic profile twice giving rise to two new antigenic types, CPV-2a and CPV-2b. These new types have replaced the original type CPV-2 in the United States of America, Europe and Japan. However, no data about the prevalence of the new antigenic types on the African continent are available. In this study, 128 recent parvovirus isolates from South Africa and Namibia were antigenically typed with type-specific monoclonal antibodies. No original CPV-2 viruses were found and its complete replacement by the new antigenic types conforms to the situation in other parts of the world. The predominant strain found in southern Africa was CPV-2b (66%), which differs from the situation in Europe and Japan where CPV-2a is the most prevalent type. Analysis of the capsid protein DNA-sequences of four selected African isolates gave no hint of a specific African parvovirus lineage.

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.

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.