Birds and viruses at a crossroad--surveillance of influenza A virus in Portuguese waterfowl.

During recent years, extensive amounts of data have become available regarding influenza A virus (IAV) in wild birds in northern Europe, while information from southern Europe is more limited. Here, we present an IAV surveillance study conducted in western Portugal 2008-2009, analyzing 1653 samples from six different species of waterfowl, with the majority of samples taken from Mallards (Anas platyrhynchos). Overall 4.4% of sampled birds were infected. The sampling results revealed a significant temporal variation in the IAV prevalence, including a pronounced peak among predominantly young birds in June, indicating that IAV circulate within breeding populations in the wetlands of western Portugal. The H10N7 and H9N2 subtypes were predominant among isolated viruses. Phylogenetic analyses of the hemagglutinin and neuraminidase sequences of H10N7, H9N2 and H11N3 virus showed that sequences from Portugal were closely related to viral sequences from Central Europe as well as to IAVs isolated in the southern parts of Africa, reflecting Portugal's position on the European-African bird migratory flyway. This study highlights the importance of Portugal as a migratory crossroad for IAV, connecting breeding stationary waterfowl with birds migrating between continents which enable transmission and spread of IAV.

Virological and serological surveillance for type A influenza in the black-legged kittiwake (Rissa tridactyla).

BACKGROUND: The epidemiology of avian influenza viruses (AIVs) in gulls is only partially known. The role of the world's most numerous gull species, the black-legged kittiwake (Rissa tridactyla), as a potential AIV reservoir species has been unclear. The prevalence of AIV and humoral response against AIV were therefore studied in a colony of apparently healthy black-legged kittiwakes breeding in a nesting cliff in the South West Barents Region of Norway (70°22' N, 31°10' E), in 2008 and 2009. RESULTS: AIVs were detected from the oropharynx and cloaca in low amounts, with prevalences of 15% and 5%, in 2008 and 2009, respectively. Direct, partial sequencing of the hemagglutinin (HA) gene revealed that the H4 subtype was present. In 2009, antibodies to influenza A virus were detected in sera from 57 of 80 adult birds. In contrast, none of the three-week-old chicks (n = 18) tested seropositive. Hemagglutination inhibition (HI) assays demonstrated that the adult kittiwakes primarily had antibodies specific to the gull-associated H13 and H16 subtypes, with antibodies to H16 being most common. CONCLUSIONS: These results support that the highly pelagic black-legged kittiwake is a reservoir of AIV. The serological findings suggest that H16 might be the main AIV subtype in the black-legged kittiwake. Further studies are needed to understand the ecology of AIV in the black-legged kittiwake and in gulls in general.

Clinical Impact of Infection with Pandemic Influenza (H1N1) 2009 Virus in Naïve Nucleus and Multiplier Pig Herds in Norway.

The Norwegian pig population has been free from influenza viruses until 2009. The pandemic influenza outbreak during the autumn 2009 provided an opportunity to study the clinical impact of this infection in an entirely naïve pig population. This paper describes the results of a case-control study on the clinical impact of pandemic influenza (H1N1) 2009 infection in the nucleus and multiplier herds in Norway. The infection spread readily and led to seroconversion of 42% of the Norwegian nucleus and multiplier herds within a year. Positive and negative herds were identified based on surveillance data from the Norwegian Veterinary Institute. Telephone interviews were conducted with pig herd owners or managers between November 2010 and January 2011. Pigs with clinical signs were reported from 40% of the case herds with varying morbidity and duration of respiratory disease and reduced reproductive performance. Clinical signs were reported in all age groups.

Pathological Findings and Distribution of Pandemic Influenza A (H1N1) 2009 Virus in Lungs from Naturally Infected Fattening Pigs in Norway.

The Norwegian pig population was considered free from influenza A virus infections until the first case of porcine pandemic influenza A (H1N1) 2009 virus infection in October 2009. Human to pig transmission of virus was suspected. Unusual lung lesions were observed in fattening pigs, with red, lobular, multifocal to coalescing consolidation, most frequently in the cranial, middle, and accessory lobes. The main histopathological findings were epithelial degeneration and necrosis, lymphocyte infiltration in the epithelial lining and lamina propria of small bronchi and bronchioles, and peribronchial and peribronchiolar lymphocyte infiltrations. Infection with pandemic influenza A (H1N1) 2009 virus was confirmed by real-time RT-PCR and immunohistochemical detection of influenza A virus nucleoprotein in the lesions. This investigation shows that natural infection with the pandemic influenza A (H1N1) 2009 virus induces lung lesions similar to lesions described in experimental studies and natural infections with other swine-adapted subtypes of influenza A viruses.

Prevalence and subtypes of influenza A viruses in wild waterfowl in Norway 2006-2007.

The prevalence of influenza A virus infection, and the distribution of different subtypes of the virus, were studied in 1529 ducks and 1213 gulls shot during ordinary hunting from August to December in two consecutive years, 2006 and 2007, in Norway. The study was based on molecular screening of cloacal and tracheal swabs, using a pan-influenza A RT-PCR. Samples found to be positive for influenza A virus were screened for the H5 subtype, using a H5 specific RT-PCR, and, if negative, further subtyped by a RT-PCR for the 3'-part of the hemagglutinin (HA) gene, encompassing almost the entire HA2, and the full-length of the neuraminidase (NA) gene, followed by sequencing and characterization. The highest prevalence (12.8%) of infection was found in dabbling ducks (Eurasian Wigeon, Common Teal and Mallard). Diving ducks (Common Goldeneye, Common Merganser, Red-breasted Merganser, Common Scoter, Common Eider and Tufted Duck) showed a lower prevalence (4.1%). In gulls (Common Gull, Herring Gull, Black-headed Gull, Lesser Black-headed Gull, Great Black-backed Gull and Kittiwake) the prevalence of influenza A virus was 6.1%. The infection prevalence peaked during October for ducks, and October/November for gulls. From the 16 hemagglutinin subtypes known to infect wild birds, 13 were detected in this study. Low pathogenic H5 was found in 17 dabbling ducks and one gull.

VPg of Potato virus A alone does not suppress RNA silencing but affects virulence of a heterologous virus.

The viral genome-linked protein (VPg) is a well-known virulence factor in potyviruses (genus Potyvirus), including Potato virus A (PVA). Its ability to suppress onset and signalling of transgene-mediated RNA silencing and accumulation of small interfering RNA (siRNA) was studied using cross-protection and Agrobacterium infiltration assays and green fluorescent protein (GFP) and PVA VPg protein-expressing transgenic Nicotiana benthamiana plants. N. benthamiana plants were also transformed with a transgene comprising the cylindrical inclusion protein (CI), nuclear inclusion protein a (NIa) and coat protein (CP) encoding regions of PVA. This transgene mRNA was expressed in the T1 progeny of the transgenic lines but all were susceptible to PVA. This result contrasted the plants transformed with the PVA P1, VPg (N-proximal part of NIa) or CP encoding regions that expressed various forms of resistance. There was little evidence for direct involvement of VPg in suppression of silencing, while other mechanisms by which VPg might interfere with transgenic resistance could not be excluded. Expression of the wild-type PVA VPg from the genome of Potato virus X (PVX, genus Potexvirus) increased symptom severity in N. benthamiana, whereas a single point mutation introduced to the VPg enhanced accumulation of the PVX chimera. These data demonstrated previously unknown virulence functions controlled by the VPg of a potyvirus.

P1- and VPg-transgenic plants show similar resistance to Potato virus A and may compromise long distance movement of the virus in plant sections expressing RNA silencing-based resistance.

Nicotiana benthamiana was transformed with P1 or VPg cistron of Potato virus A (PVA, genus Potyvirus). For both transgenes, T1 progeny displayed (i) resistance to PVA infection, (ii) susceptibility, or (iii) systemic infection followed by recovery of new leaves from PVA infection (RC), regardless of the transgene. In RC plants, fully recovered leaves contained no detectable PVA RNA, were highly resistant to challenge inoculation with PVA, and had barely detectable steady-state levels of transgene mRNA; transgene-homologous siRNA was not detected, in contrast to leaves undergoing recovery. Tops in RC plants and PVA-susceptible transgenic plants were replaced with scions from wild-type plants; only scions on the latter became PVA-infected. These findings suggest that vascular movement of PVA from lower, infected parts of RC plants was compromised in the recovered section expressing RNA silencing-based resistance, which adds a novel dimension to the current models for potyvirus movement.

Transient coexpression of individual genes encoded by the triple gene block of potato mop-top virus reveals requirements for TGBp1 trafficking.

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the "triple gene block" (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBpl to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.

Potato mop-top virus: the coat protein-encoding RNA and the gene for cysteine-rich protein are dispensable for systemic virus movement in Nicotiana benthamiana.

Full-length genomic cDNA clones of the Swedish isolate of Potato mop-top virus (PMTV) were transcribed in vitro using T7 RNA polymerase. The combination of RNA 1, 2 and 3 synthesized in the presence of m(7)GpppG cap analogue was infectious when inoculated onto Nicotiana benthamiana plants. Also, the combination of RNA 1 (encodes the viral replicase) with RNA 3 [encodes the triple gene block proteins and a small cysteine-rich protein (CRP)] was infectious and both RNAs moved systemically in N. benthamiana plants in the absence of RNA 2, which encodes the coat protein (CP). However, the yellow mosaic symptoms that typically developed following PMTV infection with all three RNAs were not observed in plants infected with RNA 1+RNA 3. Site-directed mutagenesis experiments revealed that expression of the putative CRP was not required for systemic infection and symptom induction in N. benthamiana. These data show that PMTV represents an example of a multipartite virus capable of establishing systemic infection without the CP-encoding RNA, and also without the putative CRP.

Initial infection of roots and leaves reveals different resistance phenotypes associated with coat protein gene-mediated resistance to Potato mop-top virus.

Resistance to the pomovirus Potato mop-top virus (PMTV) was studied in potato (Solanum tuberosum cv. Saturna) and Nicotiana benthamiana transformed with the coat protein (CP) gene of PMTV. The incidence of PMTV infections was reduced in tubers of the CP-transgenic potatoes grown in the field in soil infested with the viruliferous vector, Spongospora subterranea. However, in those tubers that were infected, all three virus RNAs were detected and virus titres were high. The CP-transgenic N. benthamiana plants were inoculated with PMTV using two methods. Following mechanical inoculation of leaves, no RNA 3 (the CP-encoding RNA homologous to the transgene) was detected in leaves, but in some plants low amounts of RNA 3 were detected in roots; RNA 2 was readily detected in leaves and roots of several plants. Inoculation of roots using viruliferous S. subterranea resulted in infection of roots in all plants and the three PMTV RNAs were detected. However, no systemic movement of PMTV from roots to the above-ground parts was observed, indicating a novel expression of resistance. These data indicate that the CP gene-mediated resistance to PMTV specifically restricts accumulation of PMTV RNA 3, and is more effective in leaves than roots. Furthermore, expression of resistance is different depending on whether leaves or roots are inoculated. Data do not exclude the possibility that both a protein-mediated and an RNA-mediated resistance mechanism are involved.