use in young turkeys, the most vulnerable population to NDV and aMPV diseases [2,22,38]. Turkeys vaccinated with either rLS/aMPV-A G or -B G virus had compara- ble levels of NDV-specific HI antibody response and survived the lethal dose NDV challenge without any clinical sign of disease. To properly evaluate protective efficacy of these vaccine candidates against homologous aMPV challenge, the vaccinated and control birds were challenged with pathogenic aMPV-A or -B through transmission infection to mimic natural infection. It ap- peared that the infected birds, through transmission, showed clinical signs two days later than birds challen- ged directly via IN/IO routes, indicating aMPV had a two-day incubation period while spreading through the environment. At 9 DPC with pathogenic aMPV-A or -B, the recombinant virus-vaccinated turkeys showed milder clinical signs and less virus shedding than the birds in the control groups. The lack of detectable aMPV G gene- specific antibody response and the partial protection conferred by the recombinant viruses against homolo- gous aMPV challenge suggest that the aMPV G protein is a weak antigen. Our data on the aMPV G protein, in- ducing partial protective immunity, together with the find- ings by others on immunogenicity of individual aMPV structural proteins [18,21], demonstrates that a single aMPV protein may not have the capability to induce a strong enough immune response to provide complete protection against aMPV disease. It is reasonable to spe- culate that co-expression of two or more major structural proteins of the aMPV virus, i.e. the F, G and/or M pro- teins, perhaps by the NDV vector, may be necessary to in- duce an enhanced protective immunity against aMPV in- fection.
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After receiving sequences and editing them for confirmation of obtained band, we did BLAST and results of the BLAST confirmed the presence of aMPV RNA in the samples. Bioinformatics analysis and a phylogenetic tree based on partial nucleotide sequences of the F gene shows that the detected aMPVs were classified in the subtype B (Figure 1). Detected strains were established phylogenetically in a separate branch. Similarity among Iranian strains was 99.58% -100 % (Table 1).The similarity of Iranian aMPV to type A, C and D were 77.07%,76.4% and 68.41%. The Iranian aMPV strain has the highest similarity (99.58%) to Russian type B isolates. It should be noted that negative extraction control and PCR reaction negative control were both negative that represents the accuracy of the work.
Avian metapneumovirus (aMPV) causes turkey rhinotrache- itis (TRT) and is associated with swollen head syndrome (SHS) in chickens, which is usually accompanied by secondary bacte- rial infections that increase mortality. TRT was first reported in South Africa during the early 1970s, and viruses were sub- sequently isolated in Europe, Israel, and Asia (1, 12, 23). In February 1997, the National Veterinary Services Laboratory (NVSL) (Animal and Plant Health Inspection Service, U.S. Department of Agriculture [USDA]) officially isolated aMPV from commercial turkeys in Colorado (aMPV/CO) after an outbreak of TRT the previous year. During the first 10 months of this outbreak of TRT in the United States, it was not pos- sible to detect virus serologically due to the absence of cross- reactivity of the U.S. aMPV isolates with reagents produced in Europe. An enzyme-linked immunosorbent assay (ELISA) was developed by the NVSL using inactivated aMPV/CO as an antigen, and serological evidence of aMPV infection was sub- sequently demonstrated in north-central U.S. turkey flocks (D. A. Senne, R. K. Edson, J. C. Pederson, and B. Panigrahy, unpublished data). In the United States, mortality due to aMPV infections in birds with both aMPV and bacterial infec-
Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes.
Cells, virus, and plasmids. Vero cells, BHK-21 cells, DF-1 cells, and chicken embryo fibroblasts (CEF) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Scientific, Rockford, IL) supplemented with 10% fetal bovine serum (Gibco, Invitrogen/Life Technologies, Aus- tralia) and 1% penicillin and streptomycin (Summus, Beijing, China). The aMPV/B strain aMPV/f and Newcastle disease virus (NDV), LaSota vaccine strain, were maintained in our laboratory. F genes of aMPV/B strain VCO3/60616 (GenBank accession number AB548428.1) and hu- man metapneumovirus (hMPV) Canada 97-83 strain (GenBank acces- sion number AY297749.1) were subcloned into a pCAGGS expression vector carrying a Flag tag and were named aMPV/B-F and hMPV-F, re- spectively. Monkey hepsin (Mo_hepsin; GenBank accession number XM_005588816.1), monkey TMPRSS12 (Mo_TMPRSS12; GenBank ac- cession number XM_005570851.1), human TMPRSS2 (Hu_TMPRSS2; GenBank accession number NM_005656.3), and chicken TMPRSS12 (Ch_TMPRSS12; GenBank accession number XM_424480.3) were sub- cloned into a pCAGGS vector with a hemagglutinin (HA) tag. Mutations were introduced into aMPV/B-F, hepsin, and TMPRSS12 using an In- Fusion HD cloning kit (Clontech, Mountain View, CA, USA). F, hepsin, and TMPRSS12 genes used were confirmed by DNA sequencing and Western blot analysis.
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Avian metapneumovirus (AMPV), previously known as tur- key rhinotracheitis virus or avian pneumovirus, causes an acute respiratory disease in turkeys and is also associated with “swol- len head syndrome” in chickens (10, 11, 37, 39). The virus was first isolated in South Africa in 1978 and subsequently in other parts of the world (reviewed in reference 25). AMPV was first isolated in the United States in 1996 in Colorado from com- mercial turkeys showing clinical signs of rhinotracheitis (14, 20). Subsequently, AMPV outbreaks were reported in Minne- sota, where the disease has emerged as a major economic problem for turkey farmers. Recent seroprevalence studies have indicated that the virus has also spread to other states, such as North Dakota, South Dakota, Iowa, and Wisconsin (1). AMPV is a member of the genus Metapneumovirus in the subfamily Pneumovirinae of the family Paramyxoviridae (29). The genus Metapneumovirus contains AMPV and the human metapneumovirus (HMPV). HMPV causes an acute respira- tory illness in young children and immunocompromised adults (5, 23, 36, 38). Members of the genus Metapneumovirus contain a nonsegmented, single-stranded negative-sense RNA genome with the gene order 3 ⬘ -leader-N-P-M-F-M2-SH-G-L-trailer-5 ⬘ (2, 22, 35, 40). The AMPV isolates that exist worldwide are currently classified into four subgroups, namely, subgroups A, B, C, and D. This classification is based mainly on sequence divergence observed in the attachment glycoprotein and the
Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and swollen head syndrome in chickens. Four aMPV subgroups (A-D) have been reported previously based on their genetic and antigenic differences. Evidence suggests that the live bird markets (LBMs) play an important role in the epidemiology of the avian viral diseases. A total number of 450 oropharyngeal samples from eight different species of birds (migratory and local) were collected from LBMs of Gilan province, Iran, from October to December 2016. The presence of aMPV was determined by reverse transcription polymerase chain reaction (RT-PCR) based on nucleoprotein gene. The aMPV was detected in 30.60% of the examined birds including chickens (37.00%), turkey (33.00%), Eurasian teal (25.00%), common blackbird (33.00%), and Eurasian woodcock (25.00%). Bioinformatics analysis and a phylogenetic tree based on partial nucleotide sequences of the N gene showed that the detected aMPVs were belonged to subtype B. This is the first report of aMPV in non-commercial birds in Iran. Knowledge of the frequency and types of infected birds with pneumoviruses allow a better understanding of the epidemiology of aMPV in Iran.
Hitherto, only B subtype of aMPV have been reported from Iran. The probability of circulating of other subtypes cannot be rule out due to few sequences available from Iran. It can be assumed that B subtype is dominant type of aMPV in poultry farm circulating in the country in view of the fact that other subtypes have not been detected yet. Similarly, aMPV subtype B was characterized earlier in Israeli and Jordanian poultry (15). This similarity with Brazil stains which probably reflects the policy of that country to import chicken from most major poultry producing countries in Europe and Asia.
with the F protein of aMPV subtype B, we postulated that fusion promotion for this subtype requires trypsin. In fact, trypsin is essential not only for the growth of hMPV (3, 13) but also for fusion promotion induced by its F protein (42, 43). To determine the role of trypsin in aMPV fusion, we analyzed syncytium forma- tion of the aMPV F protein following trypsin treatment. Briefly, the transfected cells were washed 4 times with PBS solution (pH 7.0), and cell monolayers were incubated with 2 ml of OPTI-MEM either with or without TPCK-trypsin (0.2 g/ml). Interestingly, small syncytia were observed for the F protein of subtype B when TPCK-trypsin was added, indicating that subtype B requires tryp- sin for cell-cell fusion (Fig. 1A). For the F proteins of aMPV sub- types A and C, syncytia were significantly enhanced and fusion occurred much earlier with the addition of trypsin (Fig. 1A). Un- fortunately, we were unable to quantitatively compare the fusion activity with or without trypsin because we had to detach the cells with trypsin in the quantitative content-mixing fusion assay. To further determine the effect of trypsin on cell-cell fusion, we de- termined the kinetics of syncytium formation of subtype A-F pro- tein by fixing the cells at different times. Large syncytia were ob- served at 14 h posttransfection for subtype A with trypsin (Fig. 2). In contrast, syncytia were not observed until 22 h posttransfection without trypsin (Fig. 2). Thus, these results demonstrate that (i) cell-cell fusion of aMPV F protein occurs at neutral pH and is independent of the G protein, and (ii) trypsin is required for aMPV B fusion and is able to enhance it but is not required for the induction of cell-cell fusion by subtypes A and C.
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Three hundred and twenty day-old chicks were randomly distributed in eight groups; each group consisted of 40 chicks per isolation unit. The vaccination treatments consisted of either a single NDV, aMPV or IBV vaccine dose, or a combination (dual or triple) of the three (Table 1). Each chick was vaccinated via oculo (50 µl) nasal (50 µl) route. Dosages received by each bird were as recommended by the manufacturers (Table 1). Following vaccination, the chicks were observed daily for clinical signs. Oropharyngeal (OP) swabs were randomly collected prior to vaccination from ten chicks and at 3, 7, 14, 21, 26 and 35 days post vaccination (dpv) from ten chicks per groups for reverse-transcriptase polymerase-chain reaction (RT-PCR) analysis. For serology, blood was collected prior to vaccination and at 21 and 35 dpv from eight chicks in each group. At 21 dpv, five chicks from each group were humanely killed and trachea samples were collected from each bird, immediately placed in aluminium foil cups containing cryo embedding compound (OCT) and frozen in liquid nitrogen (-190°C). The trachea was used for detection of CD4+, CD8+ and IgA-bearing B cells by immunohistochemistry. All samples were stored at -70 o C until processing. At 21 dpv,
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Definitive diagnosis of aMPV infection requires identification of the virus in clinical material which is most often achieved nowadays by RT-PCR. However, PCR technology is unavailable in some countries and requires transportation of samples in a safe way to specialist laboratories, international shipment of samples needs high standards of biosafety procedures (Snyder, 2002), including for aMPV. In assessing the suitability of detecting aMPV subtype A or B, or both, were reporting the results of experiments on inactivation of aMPV on FTA card, aMPV detection limits when the viruses are sampled on FTA cards, effects of different temperatures on storage of FTA cards containing aMPV, and a cross- comparison between two sampling methods (tissues directly versus tissue supernatant onto FTA cards).
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In the late 1970 in South Africa a severe pathological condition of turkeys, characterized by an acute rhinotracheitis, hit the local poultry industry heavily (24). Although many previously known respiratory pathogens could have been responsible for such a condition, no immediate diagnosis was made. Subsequent analysis pointed to an unknown viral agent, promptly named Turkey rhinotracheitis virus (TRTv), as cause of that epizootic (24). The virus was later charachterized as a negative sense, non-segmented, single stranded RNA virus closely related to mammalian respiratory syncytial viruses (178). For this reason it was classified within the Paramyxoviridae family, subfamily Pneumovirinae, genus Pneumovirus (178). More recently, following the detection of a similar virus infecting humans, TRTv was then placed in a new genus, Avian metapneumovirus (AMPV) (224). After its first detection in Africa (24) AMPV spread rapidly to Europe (7) and then to most parts of the world (50), becoming immediately a major problem for turkey production, but also for chickens, which soon proved to be susceptible to it. Losses were mainly due to a decreased bird growth rate and sometimes to high mortalities caused by secondary bacterial infections (205). Furthermore field and experimental evidence showed the viral capacity to affect egg production in laying birds, both in terms of number of laid eggs and egg quality (69, 190). Up to now AMPV control has been possible only by using live attenuated and killed vaccines (89). However, this approach has been shown to be limited, especially regarding eradication; also live vaccines have sometimes been shown to revert to virulence in turkeys (30) and as different viral subtypes are present, protection is often limited to certain strains (51). On the other hand killed vaccines appeared to be ineffective in preventing the respiratory infection, and are mainly administered to protect against egg drops (51).
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collaborative thesis research.This collaborative research consists of four research groups. The first group conducted a study of the phenomenon of rainwater falling on open and vegetated ground, using the HOTL-DI-type A model. The target subjects were first semester students and mentors were third semester students. The second group examined the phenomenon of swinging coconut trees due to wind, using the HOTL-DI-type A model. The learning subjects were students in semester III, mentored by students in semester V.The third and fourth groups conducted explorative learning research using the HOTL-DI-type B model, with the target group being the third semester students, mentored by the fifth semester students.The theme of the third group research is pottery production, and the fourth group research theme is ceramic production. Each collaborative research group consists of two students. The first student examines the process, achievements and improvement of the ability to explore objects and the other examines the democratic interaction of the mentor student group with the target student. This collaborative research was conducted from May to October 2019.The stages of the research include: (1) establishing terms of reference of collaborative research by the lecturer team, (2) recruitment of researcher students, (3) explanation of stages, substance and research activities to researcher students, (4) exploration of learning objects by researcher students, (5) exploration of learning objects by groups of students who will be prospective mentors, (6) application of the HOTL-DI model (types A and B) to target students.
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The laboratory diagnostics of the pathogenic agent of mycoplasma disease of turkeys have re- cently been carried out using molecular biology methods. PCR and its various modifications (PCR- RFLP) are important because of the higher capture rate of the bacteria in clinical section material and also due to its difficulty of cultivation (Garcia et al. 1995). Such cultivation is possible only on chicken- embryonated eggs or on selective broths, forming characteristic colonies in the shape of “fried eggs”. In summary, for the diagnosis of both types of my- coplasma the duplex PCR method, which targets the gene encoding the hemagglutinin protein could become a future alternative both for cultivation and for conventional PCR (Mardassi et al. 2005). The Table 5. Prevalence of aMPV and ILT as detected by PCR