Subtype B avian metapneumovirus resembles subtype A more closely than subtype C or human metapneumovirus with respect to the phosphoprotein,and second matrix and small hydrophobic proteins

Avian metapneumovirus (aMPV) subtype B (aMPV/B) nucleotide sequences were obtained for the phosphoprotein (P), second matrix protein (M2), and small hydrophobic protein (SH) genes. By comparison with sequences from other metapneumoviruses, aMPV/B was most similar to subtype A aMPV (aMPV/A) relative to the US subtype C isolates (aMPV/C) and human metapneumovirus (hMPV). Strictly conserved residues common to all members of the Pneumovirinae were identified in the predicted amino acid sequences of the P and M2 protein-predicted amino acid sequences. The Cys(3)-His(1) motif, thought to be important for binding zinc, was also present in the aMPV M2 predicted protein sequences. For both the P and M2-1 protein-predicted amino acid sequences, aMPV/B was most similar to aMPV/A (72 and 89% identity, respectively), having only approximately 52 and 70% identity, respectively, relative to aMPV/C and hMPV. Differences were more marked in the M2-2 proteins, subtype B having 64% identity with subtype A but < or = 25% identity with subtype C and hMPV. The A and B subtypes of aMPV had predicted amino acid sequence identities for the SH protein of 47%, and less than 20% with that of hMPV. An SH gene was not detected in the aMPV/C. Phylogenetically, aMPV/B clustered with aMPV/A, while aMPV/C grouped with hMPV.     

An improved plaque reduction virus neutralization assay for human metapneumovirus

Virus-neutralizing antibodies against human metapneumovirus (hMPV) have been shown to be important indicators for protection in experimental animal models. An improved plaque reduction virus neutralization assay to detect hMPV-specific neutralizing antibodies was designed using two prototype recombinant hMPV strains expressing green fluorescent protein (GFP). These prototypes represented each of the main antigenic variants of hMPV, because antigenic variability could have implications for vaccine development. The utility of mutations in the F gene resulting in trypsin-independent replication was also tested. Although these mutant hMPV strains could replicate in the absence of trypsin, bigger plaque size was achieved with the addition of trypsin. Insertion of the GFP gene in the genome of hMPV did not affect replication of the virus in vitro. Plaques could be detected by measuring expression of GFP after 5 days by automated scanning. Ferret, hamster, and macaque sera positive for hMPV were compared in a conventional virus neutralization assay and the plaque reduction virus neutralization assay. The results obtained with the two assays were in agreement but the improved plaque reduction virus neutralization assay was faster, more suitable for high throughput testing, and 10-fold more sensitive than the conventional virus neutralization assay.     

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses

Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.     

Detection of human metapneumovirus and respiratory syncytial virus by duplex real-time RT-PCR assay in comparison with direct fluorescent assay

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are important respiratory pathogens of small children and adults. The present study aimed to design a sensitive real-time RT-PCR assay for the detection of hRSV and hMPV in comparison with direct fluorescent assay (DFA) and to determine the incidence of hMPV and hRSV as causative agents of respiratory infections in a Finnish population. For DFA detection of hMPV antigen, four commercial antibodies were evaluated. The duplex real-time RT-PCR assay achieved a sensitivity of 10³ copies/mL of specimen for hRSV and hMPV type A viruses and 10⁴ copies/mL for type B hMPV. The detection rate of the RT-PCR assay was compared with those for DFA detection of hMPV and hRSV in analyses of 350 nasopharyngeal aspirates sent to HUSLAB, Helsinki University Hospital, for routine virus diagnostics during November 2007 to June 2008. Of the samples analyzed, 43 (12.3%) were positive for hRSV by DFA and an additional 13 specimens (3.7%) were positive for hRSV by RT-PCR. Only four samples (1.1 %) were found to be positive for hMPV RNA by RT-PCR, with two of them also positive by DFA. The duplex real-time RT-PCR assay described in the present study can therefore be applied for efficient identification of hMPV and hRSV in clinical specimens and collection of information on the epidemiology and clinical outcome of these viruses.     

Comparison of the full-length genome sequence of avian metapneumovirus subtype C with other paramyxoviruses

We determined the nucleotide (nt) sequence of the small hydrophobic (SH), attachment glycoprotein (G), and RNA polymerase (L) genes, plus the leader and trailer regions of the Colorado strain of Avian metapneumovirus subtype C (aMPV/C) in order to complete the genome sequencing. The complete genome comprised of 13,134 nucleotides, with a 40 nt leader at its 3' end and a 45 nt trailer at its 5' end. The aMPV/C L gene was the largest with 6173 nt and consisting of a single open reading frame encoding a 2005 amino acids (aa) protein. Comparison of the aMPV/C SH, G, and L nt and predicted aa sequences with those of Human metapneumoviruses (hMPV) revealed higher nt and aa sequence identities than the sequence identities between the aMPV subtypes A, B, C, and D, supporting earlier finding that aMPV/C was closer evolutionary to hMPV than the other aMPV subtypes.     

Human metapneumovirus

The human metapneumovirus (hMPV) is a new Pneumovirinae related to the avian metapneumovirus type C. hMPV genome differs from human respiratory syncytial virus (RSV) genome by the gene order and the lack of nonstructural genes. Two genetic sub-groups and four sub-types of hMPV are identified. hMPV infections evolve as regular winter outbreaks which have roughly the same size and overlaping RSV epidemics. Among hospitalized children in Caen, hMPV is detected in 9.7% of the cases after RSV (37%), rhinovirus (18%), influenza virus (14.5%), adenovirus (9%), and parainfluenza virus (5%). Most of hMPV infections are observed in children suffering from bronchiolitis, but the localization to lower respiratory tract and the severity of the disease are less frequent in comparison with RSV infections. hMPV is very difficult to isolate using cell culture. Up to now, the only way for hMPV diagnosis was the TS-CRP assays. But the recent apparition of direct antigenic tests allows us to get a fair, rapid, and economic diagnostic tool.     

Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays

A 4-tube multiplex RT-PCR (mRT-PCR), which showed higher sensitivity over conventional methods, was previously developed for the diagnosis of 14 viral pathogens of the respiratory tract. Herein the mRT-PCR was compared to the commercial Luminex mPCR-microsphere flow cytometry assay (Resplex II) which allows the detection of 12 different viruses. Eleven different viruses were identified in 91 nasopharyngeal swabs of children with acute respiratory infection, influenza A (IAV) and B, respiratory syncytial virus (RSV), human rhinovirus (hRhV), human echovirus, parainfluenza viruses (PIV) 1, 2, 3 and 4, human metapneumovirus (hMPV), and human coronavirus NL63. The results of the two techniques showed 53 and 40 positive patients by the Resplex II assay and mRT-PCR, respectively, with a concordance in 35 positive and 33 negative patients (74.7%). Individual RT-PCR tests were performed to control viruses not simultaneously detected by the two multiplex assays. The major virus misdiagnosed by mRT-PCR was IAV whereas the major viruses misdiagnosed by Resplex II were PIV1, 3 and 4. The mRT-PCR remains a simple, rapid, and specific assay for the specific detection of respiratory viruses, and can be easily implemented with standards in clinical laboratories at a low cost.     

An outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility for the elderly in Oregon

Human metapneumovirus (hMPV) was demonstrated to be responsible for an outbreak of acute respiratory tract infection with high morbidity and mortality among residents of a long-term care facility for the elderly during the late spring-summer in Oregon. Respiratory virus infections are a common cause of death in the elderly and the burden of human metapneumovirus may be underestimated. This case report stresses the importance of hMPV in causing outbreaks in long-term care facilities for the elderly. Cough and elevated temperature were common to all the resident patient cases. Six resident patient cases had hMPV laboratory confirmation of which 5 had the diagnosis of pneumonia and 4 were hospitalized. The fatality rate was 33.3% among laboratory confirmed cases and 31.3.0% among probable resident patient cases. The signs and symptoms observed in the elderly with acute respiratory infection caused by hMPV are difficult to distinguish from those associated with other respiratory viruses and direct testing for hMPV with molecular methods should be routinely pursued to prevent nosocomial infections.     

Immunochemiluminescent focus assays for the quantitation of hepatitis A virus and rotavirus in cell cultures

Two new immunological methods, the luminescent immunofocus assay (LIFA) and the luminescent immunofocus inhibition assay (LIF-IA), are described for the quantitation of cytopathic and non-cytopathic viruses propagated on cell culture monolayers. These methods use enhanced chemiluminescent detection to identify foci (luminescent immunofoci, LIF) of virus-infected cells. Viruses are propagated in susceptible cells under an agarose overlay, inactivated with ultraviolet irradiation, lifted onto nitrocellulose membranes, and probed with virus-specific monoclonal or polyclonal antibody followed by a second antibody conjugated to horseradish peroxidase. Membranes are then treated with a luminol-based detection reagent and exposed to light sensitive film for up to 10 min. The film is developed and foci appear as dark, discrete spots which are proportional to the dose of each virus. The LIFA detected both cytopathic and non-cytopathic hepatitis A viruses (HAV) and simian rotavirus. For the cytopathic HAV, the LIFA and plaque counts were comparable. The LIF-IA was developed for HAV using virus-specific antiserum which effectively attenuated LIF formation. The LIFA and LIF-IA may be completed 5 days faster than conventional radioimmunofocus assays for HAV and rotavirus and do not require the use of radiolabeled antibodies, offering safety advantages and making these techniques more adaptable for general use. Luminescent immunofocus assays should be useful for the detection and quantitation of virtually any cytopathic or non-cytopathic virus that can be propagated in monolayer cultures when virus-specific antiserum is available.     

小鼠人偏肺病毒感染模型的建立

目的 建立人偏肺病毒(hMPV)感染小鼠模型,了解病毒肺内复制规律及所致病理改变,为hMPV感染免疫病理机制研究及新型防治手段开发奠定基础.方法 BALB/c小鼠经滴鼻感染荧光标记的重组hMPV,于感染后1、3、5、7、9、16 d处死小鼠并无菌获取肺组织用于病毒分离和病理检查,改良噬斑形成法检测hMPV滴度,RT-PCR法检测hMPV mRNA表达.结果 小鼠滴鼻感染hMPV后肺组织分离到病毒;肺组织病毒滴度在感染后5 d达到高峰(5.16±1.09)×105PFU/g,感染后第9大仍能检测到病毒(2.79±1.22)×102PFU/g;感染后16 d肺组织仍可检测到hMPV mRNA;病理改变在感染后3～7 d最明显,为典型的间质性肺炎改变.结论 hMPV感染BALB/c小鼠模型建立成功,可用于hMPV感染的免疫病理机制研究.     

Nucleotide and predicted amino acid sequence-based analysis of the avian metapneumovirus type C cell attachment glycoprotein gene: phylogenetic analysis and molecular epidemiology of U.S. pneumoviruses

A serologically distinct avian metapneumovirus (aMPV) was isolated in the United States after an outbreak of turkey rhinotracheitis (TRT) in February 1997. The newly recognized U.S. virus was subsequently demonstrated to be genetically distinct from European subtypes and was designated aMPV serotype C (aMPV/C). We have determined the nucleotide sequence of the gene encoding the cell attachment glycoprotein (G) of aMPV/C (Colorado strain and three Minnesota isolates) and predicted amino acid sequence by sequencing cloned cDNAs synthesized from intracellular RNA of aMPV/C-infected cells. The nucleotide sequence comprised 1,321 nucleotides with only one predicted open reading frame encoding a protein of 435 amino acids, with a predicted M(r) of 48,840. The structural characteristics of the predicted G protein of aMPV/C were similar to those of the human respiratory syncytial virus (hRSV) attachment G protein, including two mucin-like regions (heparin-binding domains) flanking both sides of a CX3C chemokine motif present in a conserved hydrophobic pocket. Comparison of the deduced G-protein amino acid sequence of aMPV/C with those of aMPV serotypes A, B, and D, as well as hRSV revealed overall predicted amino acid sequence identities ranging from 4 to 16.5%, suggesting a distant relationship. However, G-protein sequence identities ranged from 72 to 97% when aMPV/C was compared to other members within the aMPV/C subtype or 21% for the recently identified human MPV (hMPV) G protein. Ratios of nonsynonymous to synonymous nucleotide changes were greater than one in the G gene when comparing the more recent Minnesota isolates to the original Colorado isolate. Epidemiologically, this indicates positive selection among U.S. isolates since the first outbreak of TRT in the United States.     

Changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winter-spring seasons

Summary. From 2001 through 2004, 808 pediatric patients admitted to hospital because of acute respiratory infections were examined for presence of respiratory viruses by either direct fluorescent staining using monoclonal antibodies or RT-PCR during three consecutive winter-spring seasons. On the whole, 336 (42%) patients were detected as positive for one or more respiratory viruses. The most widely circulating virus was human respiratory syncytial virus (hRSV) infecting 50% of positive patients, followed by human metapneumovirus (hMPV) found in 13% of patients, and then by influenza virus type A, human parainfluenzaviruses and coinfections. Significant variations in the circulation rate of hRSV, hMPV and influenzavirus type A were observed during the individual seasons. In addition, the circulation rates of the different types of hMPV changed yearly. In 2001–2002 and 2002–2003 hMPV circulated at a significant lower proportion than hRSV, while in 2003–2004 the circulation rates of the two viruses were closer. In conclusion, the 4 hMPV subtypes circulated yearly in Northern Italy flanking hRSV as major respiratory pathogens in the infantile patient population.     

人偏肺病毒感染人肺上皮细胞后Toll样受体的表达及其功能

目的 观察人偏肺病毒(human metapneumoviras)感染人肺上皮细胞后Toll样受体(TLR)表达变化及其信号通路的功能,探讨hMPV诱导气道炎症的部分机制.方法 hMPV感染体外培养的人肺上皮细胞株A549,检测病毒在A549细胞中的生长曲线,并通过RT-PCR,real-timeRT-PCR方法检测细胞TLR mRNA的表达,ELISA检测细胞培养上清IFN-α及TNF-α的表达.结果 (1)hMPV可在A549细胞中复制,感染后3 d达高峰10~(5.2)TCID_(50)/ml.(2)RT-PCR结果提示:hMPV感染A549细胞6 h后大部分TLR的表达均上调.(3)定量PCR结果提示:hMPV感染A549细胞后TLR3-4、TLR7-9的表达增高,且有时间依赖性,而紫外灭活的hMPV刺激细胞后TLR表达无明显变化.(4)ELISA结果提示hMPV感染后24 h,IFN-α及TNF-α的表达均明显升高.结论 人偏肺病毒感染A549细胞后可上调TLR表达,其诱导的炎性反应与部分TLR介导的信号转导途径有关.     

Prevalence of respiratory viruses, including newly identified viruses, in hospitalised children in Austria

The aim of this epidemiological study was to determine the prevalence of respiratory viruses, including new viruses, in hospitalised children in Austria. Two hundred fourteen nasopharyngeal samples from hospitalised children were tested for the presence of viruses using cell culture and PCR and/or viral antigen assays. The results revealed a parainfluenza virus 1 (PIV1) outbreak that ended right before the onset of the influenza season, with nearly no overlapping, moderate respiratory syncytial virus (RSV) activity, and only a few adenoviruses. Human metapneumovirus (hMPV) was present in 14.5% of the total samples but was detected in combination with other viruses in only five cases: with PIV1 in three cases and with RSV in two cases. There were no cases of dual infection with hMPV and flu or adenovirus. This suggests that hMPV alone is a leading cause of hospitalisation in children under 1 year of age. Interestingly, hMPV, in contrast to RSV, coincided with PIV1 but was absent during the community outbreak of the flu. Samples were also tested for Mimiviridae, a group of newly described DNA viruses that are similar to Legionella spp., replicate in water amoebae, and also have been found in alveolar cells. However, mimivirus was detected neither in respiratory samples nor in amoebae-containing water samples, indicating that this particular type of virus is either not abundant or does not contribute to paediatric respiratory illnesses.     

Detection and characterisation of human metapneumovirus from children with acute respiratory symptoms in north-west England, UK.

BACKGROUND: Human metapneumovirus (hMPV) causes a spectrum of respiratory disease ranging from trivial coryzal symptoms to fatal pneumonia, with a predilection for the very young, the immune suppressed and the frail elderly. Five distinct lineages of the virus genome have been described. OBJECTIVES: To develop and evaluate a sensitive, real-time PCR (RT-PCR) assay capable of detecting all lineages of hMPV, suitable for use in a diagnostic laboratory. STUDY DESIGN: An RT-PCR assay was developed using novel primers and dual-labelled minor-groove-binding (MGB) probes complementary to consensus sequences. The assay and two alternative assays were tested against external quality assurance (EQA) panels. 221 respiratory samples collected during 2003-2004 were screened using the new assay. hMPV positive samples were sequenced and phylogenetically analysed. RESULTS: Three genetic lineages of hMPV were detected during 2003-2004. Incidence was low (2.3%) compared to previous years. All five lineages had been present in the same community within the past 3 years. CONCLUSIONS: The new assay correctly identified more EQA samples, including those at greatest dilution, than the alternative assays and detected all five lineages. Seasonal circulation of hMPV in paediatric patients with acute respiratory symptoms is dynamic with respect to incidence and viral genotype.     

Infrared fluorescent immunofocus assay (IR-FIFA) for the quantitation of non-cytopathic and minimally cytopathic viruses

A novel method was developed for the precise quantitation of viruses using infrared fluorescent detection of foci of infection in conventional cell culture plates. In this assay, termed the infrared fluorescent immunofocus assay (IR-FIFA), appropriate cell cultures were infected with serial dilutions of hepatitis A virus (HAV) or measles virus (MV) and maintained with a semi-solid overlay for 1-5 days. Cell monolayers were fixed with formaldehyde, and then stained in succession with a primary monoclonal antibody and an Alexa Fluor 680 conjugate. Foci of infection (analogous to plaques) were detected by scanning culture plates using the Odyssey infrared imaging system and counted to determine the virus titre, expressed as focus forming units (FFU) per mL, as is done for conventional plaque assays. HAV and MV were used as models of minimally cytopathic viruses, and showed a linear dose-response between focus formation and virus dilution. Viral titres calculated using this method were comparable to conventionally used methods. The IR-FIFA was also successfully adapted to quantify duck hepatitis B virus (DHBV) as a model for a non-cytopathic virus. This simple and sensitive assay will have wide use for the quantitation of non-cytopathic and minimally cytopathic viruses.     

Efficient generation and rapid isolation via stoplight recombination of Herpes simplex viruses expressing model antigenic and immunological epitopes

Generation and isolation of recombinant herpesviruses by traditional homologous recombination methods can be a tedious, time-consuming process. Therefore, a novel stoplight recombination selection method was developed that facilitated rapid identification and purification of recombinant viruses expressing fusions of immunological epitopes with EGFP. This “traffic-light” approach provided a visual indication of the presence and purity of recombinant HSV-1 isolates by producing three identifying signals: (1) red fluorescence indicates non-recombinant viruses that should be avoided; (2) yellow fluorescence indicates cells co-infected with non-recombinant and recombinant viruses that are chosen with caution; (3) green fluorescence indicates pure recombinant isolates and to proceed with preparation of viral stocks. Adaptability of this system was demonstrated by creating three recombinant viruses that expressed model immunological epitopes. Diagnostic PCR established that the fluorescent stoplight indicators were effective at differentiating between the presence of background virus contamination and pure recombinant viruses specifying immunological epitopes. This enabled isolation of pure recombinant viral stocks that exhibited wildtype-like viral replication and cell-to-cell spread following three rounds of plaque purification. Expression of specific immunological epitopes was confirmed by western analysis, and the utility of these viruses for examining host immune responses to HSV-1 was determined by a functional T cell assay.     

Comparison of the FilmArray Respiratory Panel and Prodesse real-time PCR assays for detection of respiratory pathogens

We compared the diagnostic performance and overall respiratory pathogen detection rate of the premarket version of the FilmArray Respiratory Panel (RP) multiplex PCR assay (Idaho Technology, Inc., Salt Lake City, UT) with those of the Food and Drug Administration (FDA)-cleared Prodesse ProFlu+, ProFAST+, ProParaflu+, Pro hMPV+, and ProAdeno+ real-time PCR assays (Gen-Probe, San Diego, CA). The assays were performed on a panel of 192 nasopharyngeal-secretion specimens collected from 81 children under 1 year of age with upper respiratory tract symptoms. To resolve discordant results and confirm pathogens detected only by the larger FilmArray panel, we performed laboratory-developed real-time PCR assays. Among viruses detectable by both commercial assays (adenovirus, human metapneumovirus, influenza A virus, influenza B virus, parainfluenza viruses 1 to 3, and respiratory syncytial virus), the FilmArray and Prodesse assays showed good overall agreement (181/192 [94.3%]; kappa = 0.87; 95% CI, 0.79 to 0.94). FilmArray RP detected more parainfluenza viruses 1 and 3 than ProParaflu+ (18 versus 13) while ProAdeno+ detected more adenoviruses (11 versus 6), but these differences were not statistically significant. Additionally, FilmArray RP detected 138 pathogens (confirmed as true positives) not included in the Prodesse assays (rhinovirus [RV]/enterovirus [EV], 118; bocavirus, 8; coronavirus, 7; parainfluenza virus 4, 4; Mycoplasma pneumoniae, 1). FilmArray RP was cleared by the FDA following the completion of this study. The FDA-cleared version includes the following targets: adenovirus, coronaviruses HKU1 and NL63, human metapneumovirus (hMPV), influenza A virus (to type level only), influenza A H1 seasonal virus, influenza A H3 seasonal virus, influenza A virus H1-2009, influenza B virus, parainfluenza viruses 1 to 4, respiratory syncytial virus (RSV), and RV/EV (no differentiation). The larger panel in the FilmArray RP assay allowed the detection of additional respiratory pathogens compared to the Prodesse assays. In this population of young children with upper respiratory tract infection, RV/EV accounted for the majority of the additional pathogens detected by FilmArray RP.     

Reproducibility of swollen sinuses in broilers by experimental infection with avian metapneumovirus subtypes A and B of turkey origin and their comparative pathogenesis.

Swollen head syndrome (SHS) associated with avian metapneumovirus (aMPV) subtype A or subtype B in broilers and broiler breeders has been reported worldwide. Data about pathogenesis of aMPV subtypes A and B in broilers are scarce. It has been difficult to reproduce swollen sinuses in chickens with aMPV under experimental conditions. In the field, SHS in broilers is suspected to be induced by combined infections with different respiratory pathogens. The objectives of the present study were to compare the pathogenesis of subtypes A and B aMPV in commercial broilers and to investigate the reproducibility of clinical disease. In two repeat experiments, commercial broilers free of aMPV maternal antibodies were inoculated with aMPV subtypes A and B of turkey origin. The clinical signs such as depression, coughing, nasal exudates, and frothy eyes appeared at 4 days post inoculation, followed by swelling of periorbital sinuses at 5 days post inoculation. Higher numbers of broilers showed clinical signs in subtype-B-inoculated compared with subtype-A-inoculated groups. Seroconversion to aMPV was detectable from 10 to 11 days post inoculation. The appearance of serum aMPV enzyme-linked immunosorbent assay antibodies and the clearance of the aMPV genome coincided. Subtype B aMPV showed a broader tissue distribution and longer persistence than subtype A. Histopathological changes were observed in the respiratory tract tissues of aMPV-inoculated broilers, and also in paraocular glands, such as the Harderian and lachrymal glands. Overall, our study shows that representative strains of both aMPV turkey isolates induced lesions in the respiratory tract, accompanied by swelling of infraorbital sinuses, indicating the role of aMPV as a primary pathogen for broilers.