Avian Metapneumovirus Infection

AMPV is an enveloped virus and member of the family Pneumoviridae, genus Metapneumovirus, which currently comprises the species AMPV and HMPV. AMPV has a nonsegmented, negative-strand, single-stranded RNA genome that encodes the virus proteins nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), second matrix protein (M2), small hydrophobic protein (SH), surface glycoprotein (G), and RNA-dependent RNA polymerase (L).

The G, SH, M, N, and P proteins provide genetic heterogeneity and therefore are used for subtyping. The highly glycosylated type II membrane protein G varies the most in length and sequence, not only between subtypes but also between strains, so it is used the most frequently for classifying virus isolates.

AMPV isolates are currently grouped in subtypes A–D. Phylogenetic analysis suggests that the European subtypes A, B, and D are more closely related to each other than to subtype C, which shows more similarity on the molecular level (sequence identity, genomic organization, codon usage bias, phylogenetic location) to HMPV. Whereas European and Asian AMPV-C isolates can be grouped in one genetic sublineage, the other AMPV-C sublineage comprises the US isolates.

The circulation of AMPV subtype D was described in France, but it has not been reported there since 1980. Overall, AMPV has a wide global distribution; however, the prevalence of different subtypes varies regionally. Field strains tend to cluster according to their geographical origin. AMPV-A and AMPV-B have emerged in the US and affect primarily chickens and turkeys. Two new subtypes have been described in North America in parakeets and gulls; however, their ability to infect poultry is not yet clear. Today the most prevalent subtype in the world is AMPV-B.

Wild birds are considered natural reservoirs for AMPV. Migratory (aquatic) birds can contribute to distribution of the virus along migratory pathways; however, resident birds, especially wild Galliformes, might play a bridging role between poultry farms and potential natural reservoir species.

AMPV appears to be highly prevalent in mallard and American black ducks. Transmission of AMPV seems to depend on poultry population density, standard of hygiene, and biosecurity. Within or between poultry flocks, AMPV can be transmitted rapidly horizontally, by direct contact or by contact with contaminated material (with a morbidity rate as high as 100%).

Although AMPV is assumed to be highly contagious, this enveloped virus is rapidly destroyed after release from the host to the environment. Because AMPV affects mainly ciliated epithelial cells of the upper respiratory tract, transmission is most likely to be airborne, especially via aerosolized respiratory tract secretions.

Ciliated cells of the reproductive tract also could be target cells of AMPV. AMPV-C was isolated from eggs of experimentally infected SPF turkeys; however, the vertical transmission route appears to be short-lived and might play only a minor role in viral transmission.

Birds appear to shed AMPV for only a few days after infection. This short shedding period suggests that there is no latency or carrier status under experimental conditions. Evidence suggests that AMPV persists longer on farms (3, 4, 5, 6). Convalescent flocks can be repeatedly infected with AMPV within one fattening period.

Turkeys, chickens, and ducks can develop clinical signs, depending on which AMPV subtype causes infection. Whereas AMPV-A and AMPV-B are associated with clinical signs in chickens and turkeys, AMPV-C of the North American lineage affects mainly turkeys. AMPV-C of the Asian and European lineage affects ducks, and, less frequently, other commercial poultry, which can be subclinically infected.

Pheasants, Muscovy ducks, and captive guinea fowl can show clinical signs of AMPV infection; geese, most other duck species, and pigeons are thought to be refractory to disease. However, reports are controversial (1, 7, 8, 9), and susceptibility varies with AMPV subtype. Viral contact has also been serologically demonstrated for farmed ostriches. The US lineage of AMPV has been detected by RT-PCR in gulls, sparrows, and other wild bird species.

AMPV induces an acute, highly contagious infection of the upper respiratory tract. The main target cells are epithelial cells; however, macrophages also can carry the virus. Infection leads to clumping and loss of cilia, allowing secondary pathogens to invade. Furthermore, the virus can cause immunosuppression, leading to the replication of coinfecting pathogens. AMPV clearance coincides with the induction of AMPV antibodies and disappearance of clinical signs.

AMPV affects all age groups; however, younger birds seem more susceptible. In fattening turkeys, the virus affects primarily the upper respiratory tract. In laying hens, only mild respiratory infection, accompanied by a drop in egg production (up to 70%) and egg quality, has been observed.

Coughing associated with lower respiratory tract involvement can lead to uterine prolapse in laying turkeys.

Typical respiratory signs of AMPV in young turkeys include the following:

• serous ocular and nasal discharge (see watery ocular discharge image)

• frothy eyes (see mucopurulent ocular discharge image)

• conjunctivitis

Avian metapneumovirus infection, watery ocular discharge, turkey

Image

Courtesy of Dr. Rebecca Lindenwald.

Avian metapneumovirus infection, mucopurulent ocular discharge, turk...

Image

Courtesy of Dr. Rebecca Lindenwald.

At later stages of AMPV infection, clinical signs include mucopurulent, turbid nasal discharge; plugged nostrils; swollen infraorbital sinuses (see infected turkey flock image); and snicking, sneezing, coughing, or tracheal rales. These respiratory signs are accompanied by lethargy, anorexia, and ruffled feathers.

Avian metapneumovirus infection, turkeys

Image

Courtesy of Dr. Mirja Koy, Clinic for Poultry, University of Veterinary Medicine Hannover.

Turkeys can transmit all AMPV subtypes. Even the duck subtype C lineage replicated at low levels in turkeys under experimental conditions (10).The incubation period is 3–7 days. The mortality rate ranges from 1% to 50%, depending on age and overall constitution of the flock, as well as on the presence of secondary infections. Birds with a strong constitution that do not have secondary infections can recover within 7–10 days. In birds with secondary infections and under poor flock management conditions, however, the disease can be prolonged and exacerbated by airsacculitis, pericarditis, pneumonia, and perihepatitis, which subsequently increase condemnation rates at slaughter.

Commonly detected coinfecting bacterial pathogens include Bordetella avium, Ornithobacterium rhinotracheale, Mycoplasma spp, and Escherichia coli. Clinical signs and lesions can also be exacerbated by viral pathogens, including infectious bronchitis virus and Newcastle disease virus.

AMPV infection in chickens and pheasants is less clearly defined and might not always be associated with disease. Chickens appear more susceptible to AMPV-B than to AMPV-A. They might not show clinical signs after inoculation with AMPV-A or AMPV-C of turkey and duck lineages; however, one study reported severe respiratory signs after infection with an AMPV-C virus that formed a viral subcluster with published AMPV-C isolates (11).

AMPV is associated with swollen head syndrome in chickens. This condition is characterized by swelling of the peri- and infraorbital sinuses (see swollen head syndrome image), frothy eyes, nasal discharge, torticollis, and opisthotonos due to ear infection. Typically, < 4% of the flock is affected; however, respiratory signs can be widespread. The mortality rate is rarely > 2%; it ranges between 0.4% and 50%. In broiler breeders and commercial layers, egg production and quality are frequently affected.

Avian metapneumovirus infection, swollen head syndrome, broiler

Image

Courtesy of Dr. Ye Htut Aung, Clinic for Poultry, University of Veterinary Medicine Hannover.

AMPV-C infection of ducks can lead to respiratory signs and decreased egg production (40–85%) as well as poor eggshell quality. Clinical signs can wane in ducks after 9–12 days if no secondary infections complicate the disease.

Although it has been suggested that mice are susceptible to AMPV-C replication and develop lung lesions, in some studies, using a different AMPV strain did not lead to lesion development or viral replication (12, 13, 14, 15).

Lesions

Macroscopic lesions of AMPV infection depend on the course of infection, especially on whether secondary bacterial infections are present, and are most prominent 4–10 days after infection. Gross lesions induced after experimental infection are due to rhinitis, tracheitis, sinusitis, and airsacculitis. Infected birds can lack gross lesions.

Serous to turbid mucus can occur in the nasal cavity, nasal turbinates, trachea, and infraorbital sinuses. During the course of infection, mucus turns from clear and serous to turbid and purulent (see mucopurulent secretion image).

Avian metapneumovirus infection, mucopurulent secretion, sinus, turk...

Image

Courtesy of Dr. Arne Jung.

Nonspecific signs of inflammation, such as swelling and hyperemia of the mucosa and excessive mucus, occur in the upper respiratory tract and in the air sacs in cases of AMPV infection. If secondary bacterial infection is present, the respiratory tract contains copious inflammatory exudate. Additional signs include pneumonia, pericarditis, perihepatitis, splenomegaly, and hepatomegaly. In the reproductive tract of laying turkeys, lesions can include egg peritonitis, ovary and oviduct regression, folded shell membranes in the oviduct, and misshapen eggs. Some infected birds lack gross lesions.

Microscopic examination of the upper respiratory tract, including the secondary bronchi, during the first 2 days after AMPV infection reveals loss of cilia, increased glandular activity, congestion, and mild mononuclear infiltration of the submucosa. The most pronounced microscopic lesions are found in the upper respiratory tract mucosa or nasal turbinates, which might be the most suitable tissue for microscopic evaluation.

Harderian and lacrimal glands can also show lymphocytic infiltration and formation of lymphoid follicle-like structures in the interstitial tissue and around the secondary collecting ducts. The peak of microscopic lesion development in turkeys is expected 3–6 days after infection; it can be earlier in chickens.

• Virus detection

• Serological testing

Obtaining samples from the upper respiratory tract of birds in the early stages of the disease is critical for AMPV isolation. Especially in broiler chickens, samples should be taken before the sixth day after infection. After clinical signs are obvious, the isolation of replicating AMPV might not be successful.

The most suitable samples for AMPV detection are tracheal and choanal swabs. Tracheal organ cultures prepared from turkey or chicken embryos, or from 1- to 2-day-old chicks, are the most sensitive for primary isolation of most AMPV subtypes. Ciliostasis can occur within 7 days of inoculation with AMPV subtypes A and B but not with subtype C, or after passages.

After inoculation of 6- to 8-day-old embryonated chicken or turkey eggs via the yolk sac route, AMPV has been isolated and identified by electron microscopy, virus neutralization test, or molecular techniques. Cell cultures have not been successful for primary isolation of the virus. However, once the virus has been isolated and adapted in the systems described above, it will grow in a variety of avian and mammalian cultures, inducing a cytopathic effect.

Reverse transcription PCR (RT-PCR) assays, as well as reverse transcription quantitative real-time PCR (RT-qPCR) tests that target genes encoding the F, M, N, or G proteins of AMPV, have been developed. Some test systems are commercially available and are widely used to detect the virus in clinical samples, particularly in respiratory swabs. Samples can also be submitted on Fast Technology Analysis (FTA) cards for molecular diagnosis. Some nested RT-PCR tests have been constructed so that both the subtype and the identity of virus can be determined from the clinical sample.

The overall recommendation for AMPV detection is to apply molecular protocols targeting regions of low variability that are not subtype specific. Positive results should be followed up with additional characterization via sequencing (especially next-generation sequencing) to avoid misdiagnosis of new emerging subtypes. The high genetic heterogeneity of the gene encoding the G protein makes it suitable for strain characterization and epidemiological investigation.

These techniques have been developed into a five-plex digital droplet RT-PCR protocol (RT-ddPCR). A quick reverse transcription recombinase-aided amplification (RT-RAA) assay also enables fast detection of AMPV-C in clinical samples via isothermal amplification.

The growing amount of genome sequence data and improved access to sequencing techniques suggest that detailed characterization and molecular differentiation of isolated AMPV strains are possible, and vaccine and field strains can be identified. Thus far, however, sequencing approaches have not been able to detect host-specific variations between strains. Multiplex systems enable the detection of various coinfecting pathogens. The detection of new subtypes might require the development of new diagnostic tools.

Antigen detection tests, including immunofluorescence and immunoperoxidase assays on both fixed and unfixed tissues, have also been developed for AMPV.

Because isolation and identification of AMPV is difficult, serological assays have been developed to confirm infection in commercial chickens and turkeys. Several commercial ELISA kits are available and are commonly used; other techniques, including virus neutralization and indirect immunofluorescence tests, have also been used. Both acute and convalescent serum samples (paired serum samples from affected flocks) should be submitted for analysis.

Although ELISA systems that use either AMPV-A or AMPV-B strains as antigens detect antibodies against both of these subtypes because of some cross-reactivity, the homologous antigen should be used for efficient detection of subtype C. The subtype specificity of the applied test can result in limited or no detection of other subtypes or of new, emerging AMPV strains that do not cross-react.

Protein N might be a suitable target for the development of serological assays to detect AMPV, because it shows a high degree of identity across subtypes A, B, and D.

Paramyxoviruses—particularly the viruses that cause Newcastle disease (avian Orthoavulavirus-1 [AOAV-1]) and avian Paraavulavirus-3 (previously avian paramyxovirus-3), infectious bronchitis, and avian influenza —can cause respiratory disease and egg production problems in chickens and turkeys that closely resemble those caused by AMPV infection. These viruses can be differentiated on the basis of morphology, hemagglutinating and neuraminidase activity, and molecular characteristics.

A wide range of bacteria and Mycoplasma spp can cause clinical signs very similar to those caused by AMPV. Frequently present as secondary opportunistic pathogens, these agents can mask the presence of AMPV.

• Vaccination (live or inactivated)

• Biosecurity and management

Good management practices can markedly decrease the severity of AMPV infection, especially in turkeys. Particularly important factors affecting the outcome of the disease are ventilation, stocking density, temperature control, litter quality, and biosecurity measures.

AMPV is susceptible to lipid solvents, is stable at pH 3–9, and can be easily inactivated at temperatures > 50°C. The use of disinfectants such as quaternary ammonia, ethanol, iodophors, phenol derivatives, and bleach can decrease the viability of AMPV. Use of antimicrobials to control secondary bacterial infections has also been somewhat successful at decreasing disease severity.

Both live and inactivated vaccines are available for immunization of chickens and turkeys, and they are widely used in countries where the disease is endemic. Commercially available monovalent and multivalent inactivated vaccines protect against AMPV and other, mainly respiratory, pathogens. Studies suggest that maternal antibodies can partially interfere with vaccine virus replication and vaccination efficacy and, overall, do not provide sufficient protection against AMPV infection.

In vaccination programs targeting AMPV, the first immunization should be administered as soon as possible after hatching; it can even be given in the hatchery. It is crucial to achieve a homogenous state of immunization throughout a flock and farm by administering adequate vaccine doses to all birds.

Field evaluations indicate that AMPV escape mutants are possible, especially if the (heterologous) protection capacity of the vaccine strain is insufficient. Therefore, continued viral genome monitoringis recommended to modify preventive measures against new or reemerging subtypes and AMPV strains.

Live vaccines, which can be applied in the field by spray or via drinking water, stimulate both local respiratory and systemic immunity, and cross-protection between subtypes can occur. However, live vaccines might induce only short-lived protection, which needs especially to be considered for growing-out toms, because local immunity declines quickly. Thus, repeated vaccination of turkeys is common practice. There is, however, a risk that live vaccine strains can revert to more virulent variants. Poor vaccine application practices can lead to prolonged persistence of the vaccine strain.

For booster immunization of layer and breeder flocks, inactivated AMPV vaccines are often used to protect the reproductive tract by stimulating circulating antibodies. Whereas inactivated vaccines induce only partial protection against AMPV infection, the most efficient, homogenous, and long-lasting protection is achieved via a combined prime-boost vaccination program. This program comprises repeated priming with live, attenuated vaccines and then boosting with inactivated adjuvanted vaccines.

As experimentally shown, in ovo vaccination might also be a promising strategy for effective, early induction of an immune response. Besides live, attenuated and classic inactivated vaccines, some genetically engineered viruses, including recombinant vectored vaccines that have been designed and tested under experimental conditions, have induced partial protection and need further development.

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• Eterradossi N, Toquin D, Picault JP, Jestin V. Avian metapneumovirus. In: Brugère-Picoux J, Vaillancourt J-P, Shivaprasad HL, Venne D, Bouzouaia M, eds. Manual of Poultry Diseases; 2020:chap 20.

• Graziosi G, Lupini C, Catelli E. Disentangling the role of wild birds in avian metapneumovirus (aMPV) epidemiology: a systematic review and meta-analysis. Transbound Emerg Dis. 2022;69:3285-3299.

• Salles GBC, Pilati GVT, Muniz EC, et al. Trends and challenges in the surveillance and control of avian metapneumovirus. Viruses. 2023;15:1960.

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