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With increasing frequency, avian Metapneumovirus (aMPV) is reported to induce respiratory signs in chickens. An adequate knowledge of current aMPV prevalence among Italian broilers is lacking, with little information available on its economical and health impact on the poultry industry. In order to collect preliminary data on the epidemiological context of aMPV in broiler flocks, a survey was performed in areas of Northern Italy with high poultry density from 2014 to 2016. Upper respiratory tract swabs were collected and processed by A and B subtype-specific multiplex real-time reverse transcription PCR (RT-PCR). Samples were also screened for infectious bronchitis virus (IBV) by generic RT-PCR and sequencing. Productive data and respiratory signs were detailed where possible. The high prevalence of aMPV was confirmed in broilers older than 26 d and also attested in IBV-negative farms. All aMPV detections belonged to subtype B. Italian strain genetic variability was evaluated by the partial attachment (G) gene sequencing of selected strains and compared with contemporary turkey strains and previously published aMPV references, revealing no host specificity and the progressive evolution of this virus in Italy.

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three distinct activities contributing to the ability of HN to promote viral fusion and entry: receptor binding, receptor cleavage (neuraminidase), and activation of the fusion protein. The relationship between receptor binding and fusion triggering functions of HN are not fully understood. For Newcastle disease virus (NDV), one bifunctional site (site I) on HN′s globular head can mediate both receptor binding and neuraminidase activities, and a second site (site II) in the globular head is also capable of mediating receptor binding. The receptor analog, zanamivir, blocks receptor binding and cleavage activities of NDV HN′s site I while activating receptor binding by site II. Comparison of chimeric proteins in which the globular head of NDV HN is connected to the stalk region of either human parainfluenza virus type 3 (HPIV3) or Nipah virus receptor binding proteins indicates that receptor binding to NDV HN site II not only can activate its own fusion (F) protein but can also activate the heterotypic fusion proteins. We suggest a general model for paramyxovirus fusion activation in which receptor engagement at site II plays an active role in F activation.

Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN's receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN's F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN's F-triggering activity may have promise in vivo. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Use of real-time PCR is increasing in the diagnosis of infectious disease due to its sensitivity, specificity, and speed of detection. These characteristics make it particularly suited for the diagnosis of viral infections, like avian metapneumovirus (AMPV), for which effective control benefits from continuously updated knowledge of the epidemiological situation. Other real-time reverse transcription (RT)-PCRs have been published based on highly specific fluorescent dye-labeled probes, but they have high initial cost, complex validation, and a marked susceptibility to the genetic variability of their target sequence. With this in mind, we developed and validated a SYBR Green I-based quantitative RT-PCR for the detection of the two most prevalent AMPV subtypes (i.e., subtypes A and B). The assay demonstrated an analytical sensitivity comparable with that of a previously published real-time RT-PCR and the ability to detect RNA equivalent to approximately 0.5 infectious doses for both A and B subtypes. The high efficiency and linearity between viral titer and crossing point displayed for both subtypes make it suited for viral quantification. Optimization of reaction conditions and the implementation of melting curve analysis guaranteed the high specificity of the assay. The stable melting temperature difference between the two subtypes indicated the possibility of subtyping through melting temperature analysis. These characteristics make our assay a sensitive, specific, and rapid tool, enabling contemporaneous detection, quantification, and discrimination of AMPV subtype A and B.

Live vaccines predominantly control avian metapneumovirus (aMPV) infection in poultry flocks, but vaccine virus can be found for extended periods after application. The most frequently used aMPV vaccine in Italy, VCO3 subtype B, was shown to contain a unique Tru9I restriction endonuclease site within the amplicons produced by a commonly used aMPV diagnostic reverse transcriptase (RT)-nested polymerase chain reaction (PCR). Analysis of European and database logged subtype B aMPV sequences confirmed that the sequence occurred only in the VC03 vaccine. A subsequent RT-PCR restriction endonuclease study of field samples, collected from turkeys between 2007 and 2012, detected subtype B vaccine-derived strains in 12 of 90 samples tested that were collected from birds under 12 weeks of age.

The aim of this study was to evaluate if the exposure to Avian metapneumovirus (aMPV) and/or to Turkey hemorrhagic enteritis virus (THEV) was significant for the induction of episodes of colibacillosis in aMPV and THEV vaccinated turkeys. Colibacillosis-associated mortality was recorded and longitudinal virological studies performed in three consecutive turkey flocks reared in the same farm. aMPV and THEV diagnostic swabs and blood samples were made once a week up to 14 weeks of age. Swabs were processed by molecular techniques for viruses detection and antibody titres were evaluated. Field subtype B aMPVs were detected in all flocks at different ages of life always associated with respiratory signs and increase of colibacillosis-associated mortality. THEV has been consistently detected in all flocks since the 9th week of age. Vaccination with a single dose of the THEV commercial inactivated vaccine available in Italy seems does not protect the birds from the infection. Sequence comparison of the hexon protein of one of the THEV strains detected, and strains isolated worldwide, revealed high similarity between them. These results are consistent with the notion that the hexon protein, being the major antigenic component of the virus, is highly conserved between the strains. Results showed that field aMPV infection is directly correlated to colibacillosis-associated mortality. Less clear appears the role of THEV because the endemicity of aMPV makes difficult to evaluate its role in predisposing colibacillosis in absence of aMPV. It would be interesting to further investigate this issue through experimental trials in secure isolation conditions.

Empirically derived live avian metapneumovirus (AMPV) vaccines developed during the late 80s and early 90s have generally performed well in controlling turkey rhinotracheitis. Nonetheless, unstable attenuation was previously demonstrated in an AMPV subtype A vaccine. Until now this had not been investigated in subtype B vaccines due to lack of any similar availability of a vaccine progenitor or its sequence. The publication of the full genome sequence for the VCO3 vaccine progenitor facilitated a conclusive investigation of two AMPVs isolated from poults on a farm which had been vaccinated with VCO3 derived vaccine. Full genome sequencing of the isolates and their comparison to sequences of the vaccine and its progenitor, confirmed their vaccine origin. After determining the absence of extraneous infectious agents, one of these virus isolates was inoculated into 1-day-old turkeys in disease secure isolators and shown to cause disease with a severity similar to that caused by virulent field virus. This suggests that instability in live AMPV vaccines may be generalized and highlights the need for availability of vaccine progenitor sequences for the field assessment of all live viral vaccines.

Although avian metapneumovirus (aMPV) infection has been reported in most regions of the world, to date, only subtype B has been detected in Egypt. At the end of November 2013, dry oropharyngeal swabs were collected during an outbreak of respiratory diseases in a free-range, multi-age turkey dealer farm in Northern Upper Egypt. The clinical signs that appeared when turkeys were 3 weeks-old were characterized by ocular and nasal discharge and swelling of sinuses. aMPV of subtype A was detected by real-time reverse transcription-polymerase chain reaction. In order to confirm the results and obtain more information on the molecular characteristics of the virus, F and G protein genes were partially sequenced and compared with previously published sequences deposited in GenBank by using BLAST. Subtype of the strain was confirmed by sequencing of partial F and G protein genes. The highest percentages of identity were observed when G sequence of the Egyptian strain was compared with the sequence of an aMPV-A isolated in Nigeria (96.4 %) and when the F sequence was compared with strains isolated respectively in Italy and in UK (97.1 %). Moreover, the alignment of the sequences with commercial subtype A vaccine or vaccine-derived strains showed differences in the Egyptian strain that indicate its probable field origin. The detection of aMPV in the investigated turkey flock highlights some relevant epidemiological issues regarding the role that multi-age farms and dealers may play in perpetuating aMPV infection within and among farms. To our knowledge, this is the first report of aMPV subtype A in Egypt.

Background. The first step in infection by human parainfluenza viruses (HPIVs) is binding to the surface of respiratory epithelial cells via interaction between viral receptor-binding molecules and sialic acid-containing receptors. DAS181, a recombinant sialidase protein containing the catalytic domain of Actinomyces viscosus sialidase, removes cell surface sialic acid, and we proposed that it would inhibit HPIV infection. Methods. Depletion of sialic acid receptors by DAS181 was evaluated by lectin-binding assays. Anti-HPIV activity in cultured cell lines and in human airway epithelium was assessed by the reduction in viral genomes and/or plaque forming units on treatment. In vivo efficacy of intranasally administered DAS181 was assessed using acotton rat model. Results. DAS181-mediated desialylation led to anti-HPIV activity in cell lines and human airway epithelium. Intranasal DAS181 in cotton rats, a model for human disease, significantly curtailed infection. Conclusions. Enzymatic removal of the sialic acid moiety of HPIV receptors inhibits infection with all tested HPIV strains, both in vitro and in cotton rats. Enzyme-mediated removal of sialic acid receptors represents a novel antiviral strategy for HPIV. The results of this study raise the possibility of a broad spectrum antiviral agent for influenza virus and HPIVs. © 2010 by the Infectious Diseases Society of America. All rights reserved.

Summary Objective Respiratory viruses are a common trigger for exacerbations of chronic obstructive pulmonary disease (COPD). Human metapneumovirus (hMPV) is a paramyxovirus associated with respiratory tract infections and wheezing. Our aim was to determine whether hMPV was associated with exacerbations of COPD. Methods The study was designed as an observational cohort study carried out in a 944-bed urban teaching hospital located in New Haven, Connecticut. Between December 2002 and May 2003, patients hospitalized due to an exacerbation of COPD were identified. Nasopharyngeal specimens obtained from these patients were tested for human metapneumovirus by RT–PCR and for respiratory syncytial virus, influenza A and B, parainfluenza-1, -2, and -3 and adenovirus by a cytospin-enhanced direct immunofluorescence assay and/or viral culture. Results Fifty individuals met enrollment criteria and hMPV was identified in 6 (12%), respiratory syncytial virus in 4 (8%), influenza A in 2 (4%) and parainfluenza type 3 in 1 (2%) patients. Both A and B hMPV genotypes were identified in patients hospitalized due to exacerbations of COPD. Conclusion hMPV was frequently identified in patients hospitalized due to an exacerbation of COPD. Further studies are necessary to determine the epidemiology and the impact of hMPV in COPD patients.