Avian Paramyxovirus Type 1 from Wild Birds: Population Adaptation and Immunogenicity in Chickens.

Virus isolation rates for influenza A virus (FLUAV) and Avian paramyxovirus serotype 1 (APMV-1) from wild bird surveillance samples are lower than molecular detection rates for the specific viral genomes. The current study was conducted to examine the possibility of increased virus isolation rates from real-time reverse transcription polymerase chain reaction (real-time RT-PCR) using alternative virus isolation substrates such as embryonating duck eggs (EDEs), embryonating turkey eggs (ETEs), Madin-Darby canine kidney (MDCK) cell cultures, and African green monkey kidney (Vero) cell cultures. Rectal swabs of birds in the orders Anseriformes and Charadriiformes were tested by real-time RT-PCR for the presence of FLUAV and APMV-1 genomes, and virus isolation (VI) was attempted on all real-time RT-PCR-positive samples. Samples with threshold cycle (Ct) ≤ 37 had VI rates for FLUAV of 62.5%, 50%, 43.8%, 31.5%, and 31.5% in embryonating chicken eggs (ECEs), ETEs, EDEs, MDCK cells, and Vero cells, respectively. A higher isolation rate was seen with ECEs compared to either cell culture method, but similar isolation rates were identified between the different embryonating avian eggs. Virus isolation rates for APMV-1 on samples with real-time RT-PCR Ct ≤ 37 were 75%, 100%, 100%, 0%, and 37.5% in ECEs, ETEs, EDEs, MDCK cells, and Vero cells, respectively. Significantly higher VI rates were seen with ECEs as compared to either cell culture method for all real-time RT-PCR-positive samples. Because of the limited availability and high cost of ETEs and EDEs, the data support the continuing usage of ECEs for primary isolation of both FLUAV and APMV-1 from real-time RT-PCR-positive wild bird surveillance samples.

Avian paramyxovirus (APMV) type 4 and 6 were isolated during an avian influenza (AI) surveillance program of wild birds. This study also conducted experimental infection of wild-bird-origin APMV type 4 and 6 in specific pathogen free (SPF) chickens to study pathogenicity and transmission within domestic flocks. In addition, serological prevalence data of APMV type 4 and 6 in domestic fowls was conducted with chicken sera collected from 2007 to 2009 in order to understand infection status. The results of the animal experiment showed that APMV type 4 and 6 had the ability to infect chickens with sero-conversion and to transmit the virus from infected birds to contacted birds, but showed low pathogenicity. Serological tests revealed that APMV type 4 was widespread in the poultry industry, especially in layer flocks, but the positive rate for APMV type 6 was very low. This study concluded that wild bird-origin APMV type 4 and 6 could infect the chickens by inter-species transmission and the seroprevalence of APMV type 4 was quite high in Korean poultry. However, since almost all the chicken flocks had a high level of antibody titer against APMV type 1, there was possibility of cross reaction between APMV type 1 and 4, which made the interpretations more complicated. In order to understand infection status in the natural environment, additional study is necessary regarding the seroprevalence of APMV type 4 and 6 in the wild bird population.

The use of in situ hybridization and immunohistochemistry to study the pathogenesis of various Newcastle disease virus strains and recombinants in embryonated chicken eggs

Avian paramyxovirus type-1 (APMV-1) viruses of the lentogenic pathotypes are often isolated from wild aquatic birds and may mutate to high pathogenicity when they cross into poultry and cause debilitating Newcastle disease. This study characterised AMPV-1 isolated from fresh faecal droppings from wild aquatic birds roosting sites in Uganda. Fresh faecal samples from wild aquatic birds at several waterbodies in Uganda were collected and inoculated into 9–10-day-old embryonated chicken eggs. After isolation, the viruses were confirmed as APMV-1 by APMV-1-specific polymerase chain reaction (PCR). The cleavage site of the fusion protein gene for 24 representative isolates was sequenced and phylogenetically analysed and compared with representative isolates of the different APMV-1 genotypes in the GenBank database. In total, 711 samples were collected from different regions in the country from which 72 isolates were recovered, giving a prevalence of 10.1%. Sequence analysis of 24 isolates revealed that the isolates were all lentogenic, with the typical 111GGRQGR’L117 avirulent motif. Twenty-two isolates had similar amino acid sequences at the cleavage site, which were different from the LaSota vaccine strain by a silent nucleotide substitution T357C. Two isolates, NDV/waterfowl/Uganda/MU150/2011 and NDV/waterfowl/Uganda/MU186/2011, were different from the rest of the isolates in a single amino acid, with aspartate and alanine at positions 124 and 129, respectively. The results of this study revealed that Ugandan aquatic birds indeed harbour APMV-1 that clustered with class II genotype II strains and had limited genetic diversity.

The preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. Preliminary studies indicated that an African green monkey kidney cell line (Vero) is a suitable system for the primary isolation and cultivation of influenza A viruses (E. A. Govorkova, N. V. Kaverin, L. V. Gubareva, B. Meignier, and R. G. Webster, J. Infect. Dis. 172:250-253, 1995). We now demonstrate for the first time that Vero cells are suitable for isolation and productive replication of influenza B viruses and determine the biological and genetic properties of both influenza A and B viruses in Vero cells; additionally, we characterize the receptors on Vero cells compared with those on Madin-Darby canine kidney (MDCK) cells. Sequence analysis indicated that the hemagglutinin of Vero cell-derived influenza B viruses was identical to that of MDCK-grown counterparts but differed from that of egg-grown viruses at amino acid positions 196 to 198. Fluorescence-activated cell sorting analysis showed that although Vero cells possess predominantly alpha2,3 galactose-linked sialic acid, they are fully susceptible to infection with either human influenza A or B viruses. Moreover, all virus-specific polypeptides were synthesized in the same proportions in Vero cells as in MDCK cells. Electron microscopic and immunofluorescence studies confirmed that infected Vero cells undergo the same morphological changes as do other polarized epithelia] cells. Taken together, these results indicate that Vero cell lines could serve as an alternative host system for the cultivation of influenza A and B viruses, providing adequate quantities of either virus to meet the vaccine requirements imposed by an emerging pandemic.

Newcastle disease virus (NDV), an avian paramyxovirus type 1, is a promising vector for expression of heterologous proteins from a variety of unrelated viruses including highly pathogenic avian influenza virus (HPAIV). However, pre-existing NDV antibodies may impair vector virus replication, resulting in an inefficient immune response against the foreign antigen. A chimeric NDV-based vector with functional surface glycoproteins unrelated to NDV could overcome this problem. Therefore, an NDV vector was constructed which carries the fusion (F) and hemagglutinin-neuraminidase (HN) proteins of avian paramyxovirus type 8 (APMV-8) instead of the corresponding NDV proteins in an NDV backbone derived from the lentogenic NDV Clone 30 and a gene expressing HPAIV H5 inserted between the F and HN genes. After successful virus rescue by reverse genetics, the resulting chNDVFHN PMV8H5 was characterized in vitro and in vivo. Expression and virion incorporation of the heterologous proteins was verified by Western blot and electron microscopy. Replication of the newly generated recombinant virus was comparable to parental NDV in embryonated chicken eggs. Immunization with chNDVFHN PMV8H5 stimulated full protection against lethal HPAIV infection in chickens without as well as with maternally derived NDV antibodies. Thus, tailored NDV vector vaccines can be provided for use in the presence or absence of routine NDV vaccination.

The hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is a multifunctional protein that has receptor recognition, neuraminidase, and fusion promotion activities. Sequence analysis revealed that the HN gene of many extremely low virulence NDV strains encodes a larger open-reading frame (616 amino acids, aa) with additional 45 aa at its C-terminus when compared with that (571 aa) of virulent NDV strains. Therefore, it has been suspected that the 45 aa extension at the C-terminus of the HN may affect the NDV virulence. In this study, we generated an NDV mesogenic strain Anhinga-based recombinant virus with an HN C-terminal extension of 45 aa (rAnh-HN-ex virus) using reverse genetics technology. The biological characterization of the recombinant virus showed that the rAnh-HN-ex virus had similar growth ability to its parental virus rAnh-wt both in embryonating chicken eggs and DF-1 cells. However, the pathogenicity of this recombinant virus in embryonating chicken eggs and day-old chickens decreased, as evidenced by a longer mean death time and lower intracerebral pathogenicity index when compared with the parental virus. This is consistent with our previous finding that the recombinant LaSota virus with a 45-aa extension at its HN C-terminal was attenuated in chickens and embryonating eggs. These results suggest that the HN protein C-terminal extension may contribute to the reduced virulence in some low virulence NDV strains.

There are nine serotypes of avian paramyxovirus (APMV). Only the genome of APMV type 1 (APMV-1), also called Newcastle disease virus (NDV), has been completely sequenced. In this study, the complete nucleotide sequence of an APMV-6 serotype isolated from ducks is reported. The 16236 nt genome encodes eight proteins, nucleocapsid protein (NP), phosphoprotein (P), V protein, matrix protein (M), fusion protein (F), small hydrophobic (SH) protein, haemagglutinin-neuraminidase (HN) protein and large (L) protein, which are flanked by a 55 nt leader sequence and a 54 nt trailer sequence. Sequence comparison reveals that the protein sequences of APMV-6 are most closely related to those of APMV-1 (NDV) and -2, with sequence identities ranging from 22 to 44%. However, APMV-6 contains a gene that might encode the SH protein, which is absent in APMV-1, but present in the rubulaviruses simian virus type 5 and mumps virus. The presence of an SH gene in APMV-6 might provide a link between the evolution of APMV and rubulaviruses. Phylogenetic analysis demonstrates that APMV-6, -1, -2 (only the F and HN sequences were available for analysis) and -4 (only the HN sequences were available for analysis) all cluster into a single lineage that is distinct from other paramyxoviruses. This result suggests that APMV should constitute a new genus within the subfamily Paramyxovirinae.

We created a novel replication-incompetent vector derived from the human parainfluenza virus type 2 (hPIV2). The vector lacks the hemagglutinin-neuraminidase (HN) gene (hPIV2/∆HN) that is essential for virus attachment and release. We also generated the stable packaging cell line expressing hPIV2 HN protein derived from Vero cells (Vero-HN). As SARS-CoV-2 vaccines, we created hPIV2/∆HN-based SARS-CoV-2 vaccines expressing the receptor binding domain of the spike protein (S-RBD) of SARS-CoV-2, and S-RBD with trimer formation domain (S-RBD-FD) with or without antigen 85B (Ag85B) having strong Th1-type cytokine inducing activity. Intratracheal administration of S-RBD-FD/hPIV2/∆HN and S-RBD-FD/Ag85B/hPIV2/∆HN induced the S-RBD-specific neutralizing antibodies in sera of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2/TG), and also the sera neutralized the pseudo-typed lentivirus expressing the SARS-CoV-2 S protein. In particular, in an S-RBD-FD/Ag85B/hPIV2/∆HN-administered group, high levels of neutralizing antibodies were induced also in the bronchoalveolar lavage fluid (BALF) in the hACE2/TG mice. Further, hACE2/TG mice vaccinated with S-RBD-FD/hPIV2/∆HN or S-RBD-FD/Ag85B/hPIV2/∆HN did not exhibit weight loss and showed a high survival rate after authentic SARS-CoV-2 challenge. These results suggested that S-RBD-FD/Ag85B/hPIV2/∆HN is a vaccine candidate against SARS-CoV-2. Finally, hPIV2/∆HN or Ag85B/hPIV2/∆HN vector will be potentially applicable to vaccine development and/or human gene therapy for respiratory tract diseases.

Current vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are administered parenterally and appear to be more protective in the lower versus the upper respiratory tract. Vaccines are needed that directly stimulate immunity in the respiratory tract, as well as systemic immunity. We used avian paramyxovirus type 3 (APMV3) as an intranasal vaccine vector to express the SARS-CoV-2 spike (S) protein. A lack of pre-existing immunity in humans and attenuation by host-range restriction make APMV3 a vector of interest. The SARS-CoV-2 S protein was stabilized in its prefusion conformation by six proline substitutions (S-6P) rather than the two that are used in most vaccine candidates, providing increased stability. APMV3 expressing S-6P (APMV3/S-6P) replicated to high titers in embryonated chicken eggs and was genetically stable, whereas APMV3 expressing non-stabilized S or S-2P were unstable. In hamsters, a single intranasal dose of APMV3/S-6P induced strong serum IgG and IgA responses to the S protein and its receptor-binding domain, and strong serum neutralizing antibody responses to SARS-CoV-2 isolate WA1/2020 (lineage A). Sera from APMV3/S-6P-immunized hamsters also efficiently neutralized Alpha and Beta variants of concern. Immunized hamsters challenged with WA1/2020 did not exhibit the weight loss and lung inflammation observed in empty-vector-immunized controls; SARS-CoV-2 replication in the upper and lower respiratory tract of immunized animals was low or undetectable compared to the substantial replication in controls. Thus, a single intranasal dose of APMV3/S-6P was highly immunogenic and protective against SARS-CoV-2 challenge, suggesting that APMV3/S-6P is suitable for clinical development.

ویروس بیماری نیوکاسل (NDV) از یک گله بلدرچین ژاپنی مشکوک به بیماریND برای نخستین بار در ایران جدا سازی و شناسایی گردید. تعدادی از پرندگان گله با سنین متفاوت، مرگ و میر ناشی از بیماری را نشان دادند. بارزترین نشانه های بالینی بیماری در پرندگان، شامل کاهش اشتها، ضعف ، افت تولید تخم، اسهال و علائم عصبی بود. در معاینات کالبد گشایی، جراحات همراه خونریزی در دستگاه گوارش و بویژه روده ها و پیش معده جلب توجه نمود. نمونه برداریهای لازم جهت آزمایشهای انگل شناسی، باکتری شناسی و ویروس شناسی انجام گرفت. نتایج بررسیهای انگل شناسی و باکتری شناسی منفی بودند. ویروس تنها از نمونه های مغز جدا گردید که با کمک آزمایش ممانعت از هماگلوتیناسیون(HI) و پادتن اختصاصی NDV ، تیپ 1 پارامیگسوویروس پرندگان (APMV-1) تشخیص داده شد. برای ارزیابی بیماریزایی ویروس جدا شده، از آزمایشهای میانگین مدت زمان مرگ (MDT) در تخم مرغهای جنین دار، شاخص بیماریزایی داخل مغزی(ICPI) و شاخص بیماریزایی داخل وریدی (IVPI) در جوجه های ماکیان استفاده گردید. اندیسهای بدست آمده در آزمایشهای MDT ، IVPI و ICPI به ترتیب 40 ، 62/1 و 31/2 بودند. این شاخصها نشان دادند که ویروس1 APMV- جدا شده از لحاظ بیماریزایی در گروه سویه های ویروسی بیماری نیوکاسل با بیماریزایی بالا (ولوژنیک ) قرار میگیرد. بر پایه یافته های این تحقیق، واکسیناسیون گله های بلدرچین در ایران علیه بیماری نیوکاسل به طور اکید توصیه می گردد.

The precursor of fusion protein (Fo) in Sendai virus growth in Vero cells can be cleaved by trypsin to forms F1 and F2, which can be resolved on SDS-polyacrylamide gels. However, if disulfide bonds are preserved during electrophoresis, F1 and F2 remain linked together even after trypsin treatment (F). Sendai virus growth in embryonated chicken eggs does not contain the precursor Fo. However, an F protein was found for Sendai virus grown in eggs when disulfide bonds were preserved during electrophoresis. The hemagglutinin-neuraminidase (HN) glycoproteins also appear to be disulfide-linked to form large complexes which are observed on SDS-polyacrylamide gels of nonreduced samples.

Effective laboratory methods for identifying avian influenza virus (AIV) in wild bird populations are crucial to understanding the ecology of this pathogen. The standard method has been AIV isolation in chorioallantoic sac (CAS) of specific-pathogen-free embryonating chicken eggs (ECE), but in one study, combined use of yolk-sac (YS) and chorioallantoic membrane inoculation routes increased the number of virus isolations. In addition, cell culture for AIV isolation has been used. Most recently, real-time reverse transcriptase (RRT)-PCR has been used to detect AIV genome in surveillance samples. The purpose of this study was to develop a diagnostic decision tree that would increase AIV isolations from wild bird surveillance samples when using combinations of detection and isolation methods under our laboratory conditions. Attempts to identify AIV for 50 wild bird surveillance samples were accomplished via isolation in ECE using CAS and YS routes of inoculation, and in Madin-Darby canine kidney (MDCK) cells, and by AIV matrix gene detection using RRT-PCR. AIV was isolated from 36% of samples by CAS inoculation and 46% samples by YS inoculation using ECE, isolated from 20% of samples in MDCK cells, and detected in 54% of the samples by RRT-PCR. The AIV was isolated in ECE in 13 samples by both inoculation routes, five additional samples by allantoic, and 10 additional samples by yolk-sac inoculation, increasing the positive isolation of AIV in ECE to 56%. Allantoic inoculation and RRT-PCR detected AIV in 14 samples, with four additional samples by allantoic route alone and 13 additional samples by RRT-PCR. Our data indicate that addition of YS inoculation of ECE will increase isolation of AIV from wild bird surveillance samples. If we exclude the confirmation RT-PCR test, cost analysis for our laboratory indicates that RRT-PCR is an economical choice for screening samples before doing virus isolation in ECE if the AIV frequency is low in the samples. In contrast, isolation in ECE via CAS and YS inoculation routes without prescreening by RRT-PCR was most efficient and cost-effective if the samples had an expected high frequency of AIV.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

BackgroundThe hemagglutinin-neuraminidase (HN) protein is the major antigenic determinant of the Mumps virus (MuV) and plays an important role in the viral infectious cycle through its hemagglutination/hemadsorption (HA/HD) and neuraminidase (NA) activities. Objective: analyze the biological and immunological properties of a polypeptide derived from a highly conserved region of the HN ectodomain. Methods: a highly conserved region of the HN gene among several MuV genotypes was chosen to be cloned in a eukaryotic expression vector. The pcDNAHN176-construct was transfected into Vero cells and RNA expression was detected by RT-PCR, while the corresponding polypeptide was detected by immunofluorescence and immunochemistry techniques. The HD and NA activities were also measured. The immunogenic properties of the construct were evaluated using two systems: rabbit immunization to obtain sera for detection of the HN protein and neutralization of MuV infection, and hamster immunization to evaluate protection against MuV infection.ResultsA 567 nucleotide region from the HN gene was amplified and cloned into the plasmid pcDNA3.1. Vero cells transfected with the construct expressed a polypeptide that was recognized by a MuV-hyperimmune serum. The construct-transfected cells showed HD and NA activities. Sera from immunized rabbits in vitro neutralized two different MuV genotypes and also detected both the HN protein and the HN176 polypeptide by western blot. Hamsters immunized with the pcDNAHN176-construct and challenged with MuV showed a mild viral infection in comparison to non-immunized animals, and Th1 and Th2 cytokines were detected in them.ConclusionsThe pcDNAHN176-construct was capable of expressing a polypeptide in Vero cells that was identified by a hyperimmune serum anti Mumps virus, and these cells showed the HD and NA activities of the complete MuV HN protein. The construct also elicited a specific immune response against MuV infection in hamsters.