Maria Alessandra De Marco

A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

BackgroundAvian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results.Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC.MethodsRRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted.ResultsThe RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 108 copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR.ConclusionThe high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens.

Transmission of Hemagglutinin D222G Mutant Strain of Pandemic (H1N1) 2009 Virus

A pandemic (H1N1) 2009 virus strain carrying the D222G mutation was identified in a severely ill man and was transmitted to a household contact. Only mild illness developed in the contact, despite his obesity and diabetes. The isolated virus reacted fully with an antiserum against the pandemic vaccine strain.

Cardiac Tamponade and Heart Failure Due to Myopericarditis as a Presentation of Infection with the Pandemic H1N1 2009 Influenza A Virus

ABSTRACT We describe a fatal case of myopericarditis presenting with cardiac tamponade in a previously healthy 11-year-old child. Pandemic H1N1 2009 influenza A virus sequences were identified in throat and myocardial tissues and pericardial fluid, suggesting damage of myocardial cells directly caused by the virus.

Evidence of cross-reactive immunity to 2009 pandemic influenza A virus in workers seropositive to swine H1N1 influenza viruses circulating in Italy.

Background Pigs play a key epidemiologic role in the ecology of influenza A viruses (IAVs) emerging from animal hosts and transmitted to humans. Between 2008 and 2010, we investigated the health risk of occupational exposure to swine influenza viruses (SIVs) in Italy, during the emergence and spread of the 2009 H1N1 pandemic (H1N1pdm) virus. Methodology/Principal Findings Serum samples from 123 swine workers (SWs) and 379 control subjects (Cs), not exposed to pig herds, were tested by haemagglutination inhibition (HI) assay against selected SIVs belonging to H1N1 (swH1N1), H1N2 (swH1N2) and H3N2 (swH3N2) subtypes circulating in the study area. Potential cross-reactivity between swine and human IAVs was evaluated by testing sera against recent, pandemic and seasonal, human influenza viruses (H1N1 and H3N2 antigenic subtypes). Samples tested against swH1N1 and H1N1pdm viruses were categorized into sera collected before (n. 84 SWs; n. 234 Cs) and after (n. 39 SWs; n. 145 Cs) the pandemic peak. HI-antibody titers ≥10 were considered positive. In both pre-pandemic and post-pandemic peak subperiods, SWs showed significantly higher swH1N1 seroprevalences when compared with Cs (52.4% vs. 4.7% and 59% vs. 9.7%, respectively). Comparable HI results were obtained against H1N1pdm antigen (58.3% vs. 7.7% and 59% vs. 31.7%, respectively). No differences were found between HI seroreactivity detected in SWs and Cs against swH1N2 (33.3% vs. 40.4%) and swH3N2 (51.2 vs. 55.4%) viruses. These findings indicate the occurrence of swH1N1 transmission from pigs to Italian SWs. Conclusion/Significance A significant increase of H1N1pdm seroprevalences occurred in the post-pandemic peak subperiod in the Cs (p<0.001) whereas SWs showed no differences between the two subperiods, suggesting a possible occurrence of cross-protective immunity related to previous swH1N1 infections. These data underline the importance of risk assessment and occupational health surveillance activities aimed at early detection and control of SIVs with pandemic potential in humans.

Avian Pneumovirus infection in turkey and broiler farms in Italy: a virological, molecular and serological field survey

Abstract Avian Pneumovirus (APV) is the casual agent of Turkey Rhinotracheitis (TRT), and also causes a respiratory infection in chickens, which can result in Swollen Head Syndrome. A survey of APV infection in Italian turkey and broiler farms, in a highly populated area of Northern Italy (Verona Province) is reported. Nine turkey farms and 6 broiler farms were sampled. Sixteen birds from each group were doubly swabbed from the choanal cleft for virus isolation on tracheal organ cultures (TOC) and RT nested PCR (A and B type specific) using extracted RNA. At the same time blood samples were collected for a blocking ELISA serological assay. The broiler samples for virological assays were treated with infectious bronchitis virus (IBV) antiserum raised against serotypes prevalent in the areas sampled, thus avoiding competitive growth of IBV on TOC. Ciliostasis on TOC was taken as the indicator of the presence of the virus, and confirmation was by indirect immunofluorescence. APV was isolated and detected by RT-PCR in 19 day-old turkeys, and in 34, 42 and 48 day-old broilers. All APV strains were found to be type B. All turkeys of more than 4 weeks old were APV positive by ELISA. APV infection was found to be widely spread in the area sampled and the protocol used for virus isolation was shown to be effective. Turkey rhinotracheitis first appeared in Italy in the late 1980s, and the APV isolates involved were subsequently characterised as B types. Our results confirm APV B type to be present in Italy, but the limited findings to date need to be extended by further surveys of other Italian regions.

Reassortment ability of the 2009 pandemic H1N1 influenza virus with circulating human and avian influenza viruses: public health risk implications.

Exploring the reassortment ability of the 2009 pandemic H1N1 (A/H1N1pdm09) influenza virus with other circulating human or avian influenza viruses is the main concern related to the generation of more virulent or new variants having implications for public health. After different coinfection experiments in human A549 cells, by using the A/H1N1pdm09 virus plus one of human seasonal influenza viruses of H1N1 and H3N2 subtype or one of H11, H10, H9, H7 and H1 avian influenza viruses, several reassortant viruses were obtained. Among these, the HA of H1N1 was the main segment of human seasonal influenza virus reassorted in the A/H1N1pdm09 virus backbone. Conversely, HA and each of the three polymerase segments, alone or in combination, of the avian influenza viruses mainly reassorted in the A/H1N1pdm09 virus backbone. Of note, A/H1N1pdm09 viruses that reassorted with HA of H1N1 seasonal human or H11N6 avian viruses or carried different combination of avian origin polymerase segments, exerted a higher replication effectiveness than that of the parental viruses. These results confirm that reassortment of the A/H1N1pdm09 with circulating low pathogenic avian influenza viruses should not be misjudged in the prediction of the next pandemic.

Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian 'avian-like' H1N1 swine viruses in mice.

To examine cross‐reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian “avian‐like” (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs.

Human-Animal Interface: The Case for Influenza Interspecies Transmission.

Since the 1990s, the threat of influenza viruses to veterinary and human public health has increased. This coincides with the larger global populations of poultry, pigs, and people and with changing ecological factors. These factors include the redistribution of the human population to cities, rapid mass transportation of people and infectious agents, increased global land use, climate change, and possible changes in viral ecology that perpetuate highly pathogenic influenza viruses in the aquatic bird reservoir. The emergence of H5N1, H7N9, and H9N2 subtypes of influenza A virus and the increased genetic exchange among influenza viruses in wild aquatic birds, domestic poultry, swine, and humans pose a continuing threat to humanity. Here we consider the fundamental and practical knowledge of influenza A viruses at the human-animal interfaces to facilitate the development of novel control strategies and modified agricultural practices that will reduce or prevent interspecies transmission.

Is there a relation between genetic or social groups of mallard ducks and the circulation of low pathogenic avian influenza viruses

We investigated the circulation dynamics of low pathogenic avian influenza viruses (LPAIVs) in the mallard (Anas platyrhynchos) reservoir in Italy. In particular, we evaluated the temporal distribution of virologic findings by combining virus isolation data with a new population genetic-based study approach. Thus, during 11 consecutive sampling periods (wintering periods between 1993/94 and 2003/04), categorised into 40 sampling sub-periods, cloacal swab samples were collected from 996 wild and 16 captive-reared mallards, to be screened by RT-PCR before attempting influenza A virus isolation in embryonated eggs. Forty-eight LPAIVs were isolated from wild mallards and antigenically characterised by haemagglutination-inhibition and neuraminidase-inhibition assays. When considering LPAIV antigenic subtypes in which more than one mallard tested virus isolation positive (H1N1, n. 22; H2N3, n. 2; H5N3, n. 2; H6N5, n. 3; H6N8, n. 2; H7N3, n. 3; H11N6, n. 5), at least two birds infected with a specific HN subtype clustered within one same sampling sub-period. In the context of the novel population genetic approach, total DNA was extracted from a subset of 16 captive-reared and 65 wild ducks (2000/01 and 2001/02 sampling periods) to assess genetic diversity by amplified fragment length polymorphisms (AFLP) markers. Analyses of AFLP results showed that captive-reared mallards clustered together, whereas two main independent clusters characterised the distribution pattern of most wild mallards. Within this subset of samples, nearly identical H7N3 LPAIV strains were isolated from two wild mallards belonging to the same genetic cluster. Blood sera were also collected from the above subset of mallards and examined for antibodies to the homologous H7N3 virus strain. Four out of six wild mallards testing H7N3-seropositive by haemagglutination-inhibition assay (2001/02 period) belonged to the genetic cluster including H7N3 virus shedding ducks. Overall, our data raise the possibility of an enhanced transmission and circulation of LPAIVs in genetic or social groups of wild mallards, gathered in flocks possibly related by parentage and/or geographic origin.

Human and animal integrated influenza surveillance: a novel sampling approach for an additional transmission way in the aquatic bird reservoir

Background : infectious low pathogenic avian influenza viruses (LPaIVs) have been recently detected on feathers of wild ducks. Laboratory trial results suggested that the preen oil gland secretion, covering waterbirds’ feathers, may attract and concentrate virus particles from aIV-contaminated waters to birds’ bodies. We evaluated whether ducks can become infected by the ingestion of preen oil-associated viral particles, experimentally smeared on their plumage. In addition, we compared virologic and serologic results obtained from mallards whose feathers were experimentally infected, with those from wild mallards naturally carrying aIVs on feathers. Methods : we experimentally coated 7 mallards (anas plathyrynchos) using preen oil mixed with a LPaIV (h10n7 subtype), and housed them for 45 days with a control, uncoated duck. cloacal, oropharyngeal and feather swabs were collected from all birds and examined for aIV molecular detection and isolation. Blood samples were also taken to detect influenza specific antibodies. In addition, sera from 10 wild mallards, carrying on feathers infectious LPaIV h10n7, were examined. Results : virologic and serologic results indicated that through self- and allopreening all the birds experimentally coated with the preen oil/aIV mix and the control duck ingested viruses covering feathers and became infected. Virus isolation from feathers was up to 32 days post-coating treatment. one out of 8 wild mallards showing antibodies against type a influenza virus was seropositive for h10 subtype too. Conclusions : our experimental and field results show evidences suggesting that uninfected birds carrying viruses on their feathers, including immune ones, might play an active role in spreading aIV infection in nature. For this reason, routine aIV surveillance programs, aimed at detecting intestinal and/or respiratory viruses, should include the collection of samples, such as feather swabs, enabling the detection of viruses sticky to preened birds’ bodies....