Novel viruses in birds: Flying through the roof or is a cage needed?

Emerging viral diseases continue to have a major global impact on human beings and animals. To be able to take adequate measures in case of an outbreak of an emerging disease, rapid detection of the causative agent is a crucial first step. In this review, various aspects of virus discovery are discussed, with a special focus on recently discovered viruses in birds. Novel viruses with a potential major impact have been discovered in domestic and wild bird species in recent years using various virus discovery methods. Only a few studies report the detection of novel viruses in endangered bird species, although increased knowledge about viruses circulating in these species is important. Additional studies focusing on the exact role of a novel virus in disease and on the impact of a novel virus on bird populations are often lacking. Intensive collaboration between different disciplines is needed to obtain useful information about the role of these novel viruses.

Investigation into diseases in free-ranging ring-necked pheasants (Phasianus colchicus) in northwestern Germany during population decline with special reference to infectious pathogens.

The population of ring-necked pheasants (Phasianus colchicus) is decreasing all over Germany since the years 2008/2009. Besides impacts of habitat changes caused by current rates of land conversion, climatic influences or predators, a contribution of infectious pathogens needs also to be considered. Infectious and non-infectious diseases in free-living populations of ring-necked pheasants have been scarcely investigated so far. In the present study, carcasses of 258 deceased free-ranging pheasants of different age groups, predominantly adult pheasants, collected over a period of 4 years in the states of Lower Saxony, North Rhine-Westphalia and Schleswig-Holstein, were examined pathomorphologically, parasitologically, virologically and bacteriologically, with a focus set on infectious pathogens. A periocular and perinasal dermatitis of unknown origin was present in 62.3% of the pheasants. Additional alterations included protozoal cysts in the skeletal musculature (19.0%), hepatitis (21.7%), enteritis (18.7%), gastritis (12.6%), and pneumonia (11.7%). In single cases, neoplasms (2.6%) and mycobacteriosis (1.7%) occurred. Further findings included identification of coronaviral DNA from trachea or caecal tonsils (16.8%), siadenoviral DNA (7.6%), avian metapneumoviral RNA (6.6%), and infectious bursal disease viral RNA (3.7%). Polymerase chain reaction (PCR) on herpesvirus, avian influenza virus (AIV), paramyxovirus type 1 (PMV-1), avian encephalomyelitis virus (AEV), and chlamydia were negative. Based on the present results, there is no indication of a specific pathogen as a sole cause for population decline in adult pheasants. However, an infectious disease can still not be completely excluded as it may only affect reproduction effectivity or a certain age group of pheasants (e.g., chicks) which were not presented in the study.

Human bocavirus infection as a cause of severe acute respiratory tract infection in children.

In 2005 human bocavirus (HBoV) was discovered in respiratory tract samples of children. The role of HBoV as the single causative agent for respiratory tract infections remains unclear. Detection of HBoV in children with respiratory disease is frequently in combination with other viruses or bacteria. We set up an algorithm to study whether HBoV alone can cause severe acute respiratory tract infection (SARI) in children. The algorithm was developed to exclude cases with no other likely cause than HBoV for the need for admission to the paediatric intensive care unit (PICU) with SARI. We searched for other viruses by next-generation sequencing (NGS) in these cases and studied their HBoV viral loads. To benchmark our algorithm, the same was applied to respiratory syncytial virus (RSV)-positive patients. From our total group of 990 patients who tested positive for a respiratory virus by means of RT-PCR, HBoV and RSV were detected in 178 and 366 children admitted to our hospital. Forty-nine HBoV-positive patients and 72 RSV-positive patients were admitted to the PICU. We found seven single HBoV-infected cases with SARI admitted to PICU (7/49, 14%). They had no other detectable virus by NGS. They had much higher HBoV loads than other patients positive for HBoV. We identified 14 RSV-infected SARI patients with a single RSV infection (14/72, 19%). We conclude that our study provides strong support that HBoV can cause SARI in children in the absence of viral and bacterial co-infections.

Identification and characterization of a novel adenovirus in the cloacal bursa of gulls.

Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species.

Detection of novel divergent arenaviruses in boid snakes with inclusion body disease in The Netherlands.

Arenaviruses are bi-segmented negative-stranded RNA viruses, which were until recently only detected in rodents and humans. Now highly divergent arenaviruses have been identified in boid snakes with inclusion body disease (IBD). Here, we describe the identification of a new species and variants of the highly divergent arenaviruses, which were detected in tissues of captive boid snakes with IBD in The Netherlands by next-generation sequencing. Phylogenetic analysis of the complete sequence of the open reading frames of the four predicted proteins of one of the detected viruses revealed that this virus was most closely related to the recently identified Golden Gate virus, while considerable sequence differences were observed between the highly divergent arenaviruses detected in this study. These findings add to the recent identification of the highly divergent arenaviruses in boid snakes with IBD in the United States and indicate that these viruses also circulate among boid snakes in Europe.

Use of influenza A viruses expressing reporter genes to assess the frequency of double infections in vitro.

Exchange of gene segments between mammalian and avian influenza A viruses may lead to the emergence of potential pandemic influenza viruses. Since co-infection of single cells with two viruses is a prerequisite for reassortment to take place, we assessed frequencies of double-infection in vitro using influenza A/H5N1 and A/H1N1 viruses expressing the reporter genes eGFP or mCherry. Double-infected A549 and Madin-Darby canine kidney cells were detected by confocal microscopy and flow cytometry.

Characterization of the human CD8⁺ T cell response following infection with 2009 pandemic influenza H1N1 virus.

The 2009 H1N1 influenza pandemic provided an opportunity to study human virus-specific T cell responses after infection with a novel influenza virus against which limited humoral immunity existed in the population. Here we describe the magnitude, kinetics, and nature of the virus-specific T cell response using intracellular gamma interferon (IFN-γ) staining and fluorochrome-labeled major histocompatibility complex (MHC) class I-peptide complexes. We demonstrate that influenza virus-infected patients develop recall T cell responses that peak within 1 week postinfection and that contract rapidly. In particular, effector cell frequencies declined rapidly postinfection in favor of relatively larger proportions of central memory cells.

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets.

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.

Prevalence of antibodies against seasonal influenza A and B viruses in children in Netherlands.

To gain insight into the age at which children become infected with influenza viruses for the first time, we analyzed the seroprevalence of antibodies against influenza viruses in children 0 to 7 years of age in the Netherlands. Serum samples were collected during a cross-sectional population-based study in 2006 and 2007 and were tested for the presence of antibodies against influenza A/H1N1, A/H3N2, and B viruses representative of viruses present in previous influenza seasons using the hemagglutination inhibition assay. The seroprevalence of antibodies to influenza virus was higher in children 1 to 6 months of age than in children 7 to 12 months of age, which likely reflects the presence of maternally derived antibodies. The proportion of study subjects >1 year of age with detectable antibodies against influenza viruses gradually increased with age until they reached the age of 6 years, when they all had antibodies to at least one influenza A virus. These findings may have implications for the development of vaccination strategies aiming at the protection of young children against seasonal and/or pandemic influenza virus infection.

Inter-observer variability of visual analysis of "stress"-only adenosine first-pass myocardial perfusion imaging in relation to clinical experience and reading criteria.

To assess the inter-observer agreement of adenosine "stress"-only visual analysis of perfusion MR images in relation to experience and reading criteria. 106 adenosine perfusion MR examinations out of 350, 46 consecutive positive examinations and 60 randomly selected negative examinations were visually analysed by three individual readers (two residents and a technician) with different levels of experience. Readings (blinded for any information) were compared with the reading of an expert radiologist. After a month the examinations were presented again (randomly) without knowledge regarding the first readings. This time readings were performed with the systematical use of reading criteria. Agreement with the expert reading was good for the most experienced resident (k = 0.88). Kappa was 0.48 for the least experienced, and 0.57 for the technician. After the second systematical reading inter-observer agreement increased to 0.9, 0.68 and 0.77 respectively. Overall kappa increased from 0.59 to 0.71. The use of reading criteria significantly improved the performance of the least experienced reader (P = 0.01). Visual analysis of adenosine "stress"-only first-pass perfusion MR images has moderate to very good agreement. Performance is experience related, but the systematic use of reading criteria significantly increased performance for the least experienced observer.

Redundancy of the influenza A virus-specific cytotoxic T lymphocyte response in HLA-B*2705 transgenic mice limits the impact of a mutation in the immunodominant NP(383-391) epitope on influenza pathogenesis.

During the 1993-1994 flu season, influenza A/H3N2 viruses emerged with an amino acid substitution (R384G) at the anchor residue of the HLA-B*2705 restricted NP(383-391) epitope located in the nucleoprotein (NP). The R384G substitution reached fixation rapidly and abrogated recognition of A/H3N2 viruses by NP(383-391)-specific CD8+ T cytotoxic T lymphocytes (CTL) completely. To test the impact of the R384G substitution in the immunodominant NP(383-391) epitope in vivo, influenza A viruses that differ at position 384 of the NP only were generated by reverse genetics. These viruses with an arginin (384R) or a glycin (384G) at position 384 were used to inoculate HLA-B*2705-transgenic mice and C57Bl/6 mice. Infection of naïve C57Bl/6 and HLA-B*2705 mice with influenza virus containing the NP(383-391) epitope (384R) caused more weight loss compared to infection with the virus without the epitope (384G). In contrast, HLA-B*2705 transgenic mice primed for a secondary CTL response by infection with a heterosubtypic influenza A virus, did not display this difference in virulence and the outcome of infection with the 384R virus was somewhat reduced. This phenotype of the 384R-virus was not observed in primed C57Bl/6 mice lacking HLA-B*2705. The relative reduction of weight loss after infection of primed HLA-B*2705+ mice with the 384R virus correlated with the CTL response to the NP(383-391). However, no differences were observed in the kinetics of viral clearance between the two viruses in immune HLA-B*2705+ mice, which may be attributed at least partially to CTL responses to other HLA-B*2705 restricted epitopes that were similar in magnitude.

A single immunization with CoVaccine HT-adjuvanted H5N1 influenza virus vaccine induces protective cellular and humoral immune responses in ferrets.

Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 microg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.

The novel adjuvant CoVaccineHT increases the immunogenicity of cell-culture derived influenza A/H5N1 vaccine and induces the maturation of murine and human dendritic cells in vitro.

A candidate influenza H5N1 vaccine based on cell-culture-derived whole inactivated virus and the novel adjuvant CoVaccineHT was evaluated in vitro and in vivo. To this end, mice were vaccinated with the whole inactivated influenza A/H5N1 virus vaccine with and without CoVaccineHT and virus-specific antibody and cellular immune responses were assessed. The addition of CoVaccineHT increased virus specific primary and secondary antibody responses against the homologous and an antigenically distinct heterologous influenza A/H5N1 strain. The superior antibody responses induced with the CoVaccineHT-adjuvanted vaccine correlated with the magnitude of the virus-specific CD4+ T helper cell responses. CoVaccineHT did not have an effect on the magnitude of the CD8+ T cell response. In vitro, CoVaccineHT upregulated the expression of co-stimulatory molecules both on mouse and human dendritic cells and induced the secretion of pro-inflammatory cytokines TNF-alpha, IL-6, IL-1beta and IL-12p70 in mouse- and IL-6 in human dendritic cells. Inhibition experiments indicated that the effect of CoVaccineHT is mediated through TLR4 signaling. These data suggest that CoVaccineHT also will increase the immunogenicity of an influenza A/H5N1 vaccine in humans.

Infection of mice with a human influenza A/H3N2 virus induces protective immunity against lethal infection with influenza A/H5N1 virus.

The transmission of highly pathogenic avian influenza (HPAI) A viruses of the H5N1 subtype from poultry to man and the high case fatality rate fuels the fear for a pandemic outbreak caused by these viruses. However, prior infections with seasonal influenza A/H1N1 and A/H3N2 viruses induce heterosubtypic immunity that could afford a certain degree of protection against infection with the HPAI A/H5N1 viruses, which are distantly related to the human influenza A viruses. To assess the protective efficacy of such heterosubtypic immunity mice were infected with human influenza virus A/Hong Kong/2/68 (H3N2) 4 weeks prior to a lethal infection with HPAI virus A/Indonesia/5/05 (H5N1). Prior infection with influenza virus A/Hong Kong/2/68 reduced clinical signs, body weight loss, mortality and virus replication in the lungs as compared to naive mice infected with HPAI virus A/Indonesia/5/05. Priming by infection with respiratory syncytial virus, a non-related virus did not have a beneficial effect on the outcome of A/H5N1 infections, indicating that adaptive immune responses were responsible for the protective effect. In mice primed by infection with influenza A/H3N2 virus cytotoxic T lymphocytes (CTL) specific for NP(366-374) epitope ASNENMDAM and PA(224-232) SCLENFRAYV were observed. A small proportion of these CTL was cross-reactive with the peptide variant derived from the influenza A/H5N1 virus (ASNENMEVM and SSLENFRAYV respectively) and upon challenge infection with the influenza A/H5N1 virus cross-reactive CTL were selectively expanded. These CTL, in addition to those directed to conserved epitopes, shared by the influenza A/H3N2 and A/H5N1 viruses, most likely contributed to accelerated clearance of the influenza A/H5N1 virus infection. Although also other arms of the adaptive immune response may contribute to heterosubtypic immunity, the induction of virus-specific CTL may be an attractive target for development of broad protective vaccines. Furthermore the existence of pre-existing heterosubtypic immunity may dampen the impact a future influenza pandemic may have.

[An update on viral diseases of the dog and cat]. / Recente ontwikkelingen op het gebied van virusziekten bij de hond en de kat.

In this review, recent developments in the field of viral diseases of the dog and the cat are discussed. In the dog, infection with the coronavirus type 2 is associated with respiratory signs, while infection of a highly pathogenic strain of the coronavirus type 1 has been identified as the cause of mortality in puppies. A new strain of the canine parvovirus is identified, from which the pathogenicity is not yet completely clarified. Infection with West Nile virus is associated with progressive neurological disease and subclinical infections in dogs. Infection with equine influenza A (H3N8) or a highly related influenza virus can cause severe respiratory disease and mortality in greyhounds and other dogs. Infection with avian influenza A (H5N1) can cause disease and mortality in cats and is mostly subclinical in dogs. A number of outbreaks of highly virulent strains of the calicivirus in cats have been described.

Primary influenza A virus infection induces cross-protective immunity against a lethal infection with a heterosubtypic virus strain in mice.

In order to assess the level of protection against a lethal influenza virus infection provided by a primary infection with a virus strain of another subtype, C57BL/6 mice were infected with the sublethal influenza virus X-31 (H3N2) and subsequently challenged with the lethal strain A/PR/8/34 (H1N1). The outcome of the challenge infection was compared with that in mice that did not experience an infection with influenza virus X-31 prior to the challenge infection. The X-31 experienced mice cleared the infection with influenza virus A/PR/8/34 in an accelerated fashion, displayed less clinical signs and a reduction of lesions in the lungs resulting in improved survival rates of these mice compared to the naive mice. The improved outcome of the challenge infection with influenza virus A/PR/8/34 in the X-31 experienced mice correlated with priming for anamnestic virus-specific CD8(+) cytotoxic T lymphocyte (CTL) responses as was demonstrated by the detection of CTL specific for the H-2D(b) restricted NP(366-374) epitope that was shared by the influenza viruses X-31 and A/PR/8/34. Thus previous exposure to influenza A viruses affords partial protection against infection in the absence of virus-neutralizing antibodies specific for the hemagglutinin and the neuraminidase. The implications of these observations are discussed in the light of the current pandemic threat and development of vaccines that aim at the induction of virus-specific CTL.