Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica).

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India.

Complete genome sequence of a novel avian paramyxovirus isolated from wild birds in South Korea.

A novel avian paramyxovirus (APMV), Cheonsu1510, was isolated from wild bird feces in South Korea and serologically and genetically characterized. In hemagglutination inhibition tests, antiserum against Cheonsu1510 showed low reactivity with other APMVs and vice versa. The complete genome of Cheonsu1510 comprised 15,408 nucleotides, contained six open reading frames (3'-N-P-M-F-HN-L-5'), and showed low sequence identity to other APMVs (< 63%) and a unique genomic composition. Phylogenetic analysis revealed that Cheonsu1510 was related to but distinct from APMV-1, -9, and -15. These results suggest that Cheonsu1510 represents a new APMV serotype, APMV-17.

Assessment of contemporary genetic diversity and inter-taxa/inter-region exchange of avian paramyxovirus serotype 1 in wild birds sampled in North America.

BACKGROUND: Avian paramyxovirus serotype 1 (APMV-1) viruses are globally distributed, infect wild, peridomestic, and domestic birds, and sometimes lead to outbreaks of disease. Thus, the maintenance, evolution, and spread of APMV-1 viruses are relevant to avian health. METHODS: In this study we sequenced the fusion gene from 58 APMV-1 isolates recovered from thirteen species of wild birds sampled throughout the USA during 2007-2014. We analyzed sequence information with previously reported data in order to assess contemporary genetic diversity and inter-taxa/inter-region exchange of APMV-1 in wild birds sampled in North America. RESULTS: Our results suggest that wild birds maintain previously undescribed genetic diversity of APMV-1; however, such diversity is unlikely to be pathogenic to domestic poultry. Phylogenetic analyses revealed that APMV-1 diversity detected in wild birds of North America has been found in birds belonging to numerous taxonomic host orders and within hosts inhabiting multiple geographic regions suggesting some level of viral exchange. However, our results also provide statistical support for associations between phylogenetic tree topology and host taxonomic order/region of sample origin which supports restricted exchange among taxa and geographical regions of North America for some APMV-1 sub-genotypes. CONCLUSIONS: We identify previously unrecognized genetic diversity of APMV-1 in wild birds in North America which is likely a function of continued viral evolution in reservoir hosts. We did not, however, find support for the emergence or maintenance of APMV-1 strains predicted to be pathogenic to poultry in wild birds of North America outside of the order Suliformes (i.e., cormorants). Furthermore, genetic evidence suggests that ecological drivers or other mechanisms may restrict viral exchange among taxa and regions of North America. Additional and more systematic sampling for APMV-1 in North America would likely provide further inference on viral dynamics for this infectious agent in wild bird populations.

Co-circulation of genetically distinct groups of avian paramyxovirus type 1 in pigeon Newcastle disease in Iran.

Pigeons are considered as one of the major natural reservoirs in the epidemiology of Newcastle disease (ND). In this study, the partial sequence of fusion protein gene of 17 pigeon-origin ND viruses (NDVs) isolated during 2012-2013 in Iran was analysed. Since the studied isolates showed F0 protein cleavage sites compatible with velogenic NDVs, all were considered as virulent NDVs. Two isolates carried 112RRQKRF117 as the cleavage site motif, whereas the rest demonstrated 112KRQKRF117 motif which just recently has been reported among Iranian virulent NDVs. Phylogenetic analysis divided all these diverse isolates in two distinct clusters within class II genotype VI. Based on the partial fusion protein gene sequence, 15 out of 17 isolates showed the highest genetic identity to subgenotype VIb/2 and the other two isolates were placed in a distinct genetic group of genotype VI. Based on recent findings, at least two different sublineages of genotype VI are causing the ND outbreaks in the pigeon population and are circulating simultaneously along with virulent NDVs of genotype VII in various species in Iran. The continuing circulation of a diverse group of virulent NDVs as an enzootic in widespread species such as pigeon can cause outbreaks in commercial poultry flocks and also failure in controlling programmes. Therefore, the constant monitoring and awareness of the virus characteristics should be considered in controlling programmes against ND in Iran.

Six-year surveillance of Newcastle disease virus in wild birds in north-eastern Spain (Catalonia).

Given that Newcastle disease (ND) is one of the major threats for the poultry industry, testing of Newcastle disease virus (NDV) has been carried out since 2010 in cases of mortality in wild birds (passive surveillance) in Catalonia. The objective is to provide an early warning system to prevent the infection of poultry. Since 2010, 35 episodes of mortality in wild birds were attributed to NDV infection. Throughout this period there was a progressive expansion of NDV to new areas, with an increase in the episodes of mortality, although it is not clear whether they were the result of the spread of the virus, or of the improvement of the surveillance. Phylogenetic analyses indicate that two distinct sublineages of NDV, 4a and 4b, were circulating in Catalonia. Both sublineages seem to be endemic in the wild bird population, affecting mainly Eurasian-collared doves, with a clear pattern in relation to its spatial distribution (coincident with the distribution of this species), and its temporal distribution (with the majority of cases between September and February). So far, endemicity in wild birds has not resulted in ND outbreaks in poultry. However, there are still many uncertainties about, for example, whether NDV may expand to new areas of Catalonia (with higher poultry density), or about the threat that the apparently more novel sublineage 4a may represent. Hence, efforts should be made so that measures to prevent infection of poultry farms (particularly in high-risk areas and periods) are encouraged, and surveillance is maintained.

Neuropathogenic Capacity of Lentogenic, Mesogenic, and Velogenic Newcastle Disease Virus Strains in Day-Old Chickens.

Strains of Newcastle disease virus (NDV) have different abilities to elicit neurologic signs. To determine the capacity of different NDV strains to replicate and cause lesions in the brain, independently of their peripheral replication, 1-day-old chickens were inoculated in the subdural space with 7 NDV strains of different virulence (4 velogenic, 2 mesogenic, 1 lentogenic). Velogenic strains induced severe necrotizing and heterophilic ventriculitis and meningitis, as well as edema of the neuroparenchyma, and replicated extensively in the nervous tissue by day 2 postinfection, as demonstrated by immunohistochemistry, when all infected birds died. Clinical signs, microscopic lesions, and viral replication were delayed (days 3 and 4 postinfection) with mesogenic strains. Velogenic and mesogenic NDV strains replicated mainly in neurons, and immunolabeling was first detected in surface-oriented areas (periventricular and submeningeal), possibly as a reflection of the inoculation route. The lentogenic NDV strain did not cause death of infected birds; replication was confined to the epithelium of the ependyma and choroid plexuses; and lesions consisted of lymphoid aggregates limited to the choroid plexuses. Results show that extensive NDV replication in the brain is typical of velogenic and mesogenic, but not lentogenic, NDV strains. In addition, this study suggests that differences in the rate of NDV replication in nervous tissue, not differences in neurotropism, differentiate velogenic from mesogenic NDV strains. This study indicates that intracerebral inoculation might be used as an effective method to study the mechanisms of NDV neuropathogenesis.

Simultaneous detection of major pome fruit viruses and a viroid.

A rapid and sensitive two-step RT-PCR protocol for simultaneous detection of major apple viruses, namely Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd), was developed. Five specific primer pairs were tested and confirmed for these viruses and viroid together in a single tube, giving amplicons of ~198, ~330, ~370, ~547 and ~645 bp corresponding to ASGV, ASSVd, ASPV, ApMV and ACLSV, respectively. Using a guanidinium-based extraction buffer along with a commercial kit resulted in better quality RNA as compared to kit, suited for multiplex RT-PCR. A rapid CTAB method for RNA isolation from apple tissue was developed, which produce good yield and saves time. To the best of our knowledge, this is the first report on the simultaneous detection of five pathogens (four viruses and a viroid) from apple with NADH dehydrogenase subunit 5 (nad5) as an internal control.

Genetic Characterization of Avian Paramyxovirus Isolated from Wild Waterfowl in Korea between 2015 and 2021.

Avian paramyxoviruses (APMVs) are often carried by wild waterfowl, and the wild waterfowl may play an important role in the maintenance and spread of these viruses. In this study, we investigated APMVs in the population of migratory wild waterfowl from 2015 to 2021 in Korea and analyzed their genetic characteristics. Fourteen viruses were isolated and subsequently identified as APMV-1 (n = 13) and APMV-13 (n = 1). Phylogenetic analysis of the full fusion gene of 13 APMV-1 isolates showed that 10 APMV-1 isolates belonged to the class II sub-genotype I.2, which was epidemiologically linked to viruses from the Eurasian continent, and 3 viruses belonged to class I, which linked to viruses from the USA. The APMV-13 isolates from wild geese in this study were highly homology to the virus isolated from China. Sequence analysis of 14 isolates showed that all isolates had a typical lentogenic motif at the cleavage site. In summary, we identified the wild species likely to be infected with APMV and our data suggest possible intercontinental transmission of APMV by wild waterfowl. Our current study also provides the first evidence for the presence of class I of APMV-1 and APMV-13 in wild waterfowl surveyed in Korea.

Avian Influenza Virus and Avian Paramyxoviruses in Wild Waterfowl of the Western Coast of the Caspian Sea (2017-2020).

The flyways of many different wild waterfowl pass through the Caspian Sea region. The western coast of the middle Caspian Sea is an area with many wetlands, where wintering grounds with large concentrations of birds are located. It is known that wild waterfowl are a natural reservoir of the influenza A virus. In the mid-2000s, in the north of this region, the mass deaths of swans, gulls, and pelicans from high pathogenicity avian influenza virus (HPAIV) were noted. At present, there is still little known about the presence of avian influenza virus (AIVs) and different avian paramyxoviruses (APMVs) in the region's waterfowl bird populations. Here, we report the results of monitoring these viruses in the wild waterfowl of the western coast of the middle Caspian Sea from 2017 to 2020. Samples from 1438 individuals of 26 bird species of 7 orders were collected, from which 21 strains of AIV were isolated, amounting to a 1.46% isolation rate of the total number of samples analyzed (none of these birds exhibited external signs of disease). The following subtypes were determined and whole-genome nucleotide sequences of the isolated strains were obtained: H1N1 (n = 2), H3N8 (n = 8), H4N6 (n = 2), H7N3 (n = 2), H8N4 (n = 1), H10N5 (n = 1), and H12N5 (n = 1). No high pathogenicity influenza virus H5 subtype was detected. Phylogenetic analysis of AIV genomes did not reveal any specific pattern for viruses in the Caspian Sea region, showing that all segments belong to the Eurasian clades of classic avian-like influenza viruses. We also did not find the amino acid substitutions in the polymerase complex (PA, PB1, and PB2) that are critical for the increase in virulence or adaptation to mammals. In total, 23 hemagglutinating viruses not related to influenza A virus were also isolated, of which 15 belonged to avian paramyxoviruses. We were able to sequence 12 avian paramyxoviruses of three species, as follows: Newcastle disease virus (n = 4); Avian paramyxovirus 4 (n = 5); and Avian paramyxovirus 6 (n = 3). In the Russian Federation, the Newcastle disease virus of the VII.1.1 sub-genotype was first isolated from a wild bird (common pheasant) in the Caspian Sea region. The five avian paramyxovirus 4 isolates obtained belonged to the common clade in Genotype I, whereas phylogenetic analysis of three isolates of Avian paramyxovirus 6 showed that two isolates, isolated in 2017, belonged to Genotype I and that an isolate identified in 2020 belonged to Genotype II. The continued regular monitoring of AIVs and APMVs, the obtaining of data on the biological properties of isolated strains, and the accumulation of information on virus host species will allow for the adequate planning of epidemiological measures, suggest the most likely routes of spread of the virus, and assist in the prediction of the introduction of the viruses in the western coastal region of the middle Caspian Sea.

Molecular Detection and Characterization of Newcastle Disease Virus from Chickens in Mid-Rift Valley and Central Part of Ethiopia.

Background: Newcastle disease (ND) is a highly infectious poultry disease that causes major economic losses worldwide. The disease is caused by Newcastle Disease Virus (NDV) and early detection and identification of the viral strain is essential. Having knowledge of the NDV strain genotype that circulates in some regions would help in designing an effective vaccine to control the disease. In this regard, there is little information on NDV strain in chickens in mid Rift Valley and the central part of Ethiopia. Therefore, the purpose of this study was to detect and characterize NDV strain genotype from chickens in mid-Rift Valley and the central part of Ethiopia and test whether this NDV strain genotype matches the vaccine strain currently used in the study area. Methods: A total of 98 samples: 78 (tracheal and cloacal) swabs from chicken pools of five and 20 tissue samples were collected. To detect NDV strain, conserved region of the virus Matrix (M) gene was amplified by qRT-PCR. To characterize NDV strain genotypes, M-gene positive samples were specifically re-amplified by conventional PCR targeting the Fusion (F) gene region and sequenced by Sanger method. Results: 13.26% of tested samples were positive for NDV strain in the study area with statistically significant difference (P<0.05) among the study sites. Further characterization of the F genes from NDV strain isolates by phylogenetic analysis indicated that one field isolate clustered with genotype VII whereas three of the isolates clustered to genotype I, II, and III. The isolate of the current NDV strain vaccine in use in the study area clustered with genotype II. Conclusion: The current study indicates the existence of different NDV strain genotype from that of the vaccine strain currently used. Even though large-scale characterization of several isolates is required at national level, the current study laid baseline information for the existence of variations between field NDV strain genotype and vaccine strain currently used against ND in the country.

Development of one step colorimetric RT-LAMP assays for rapid detection of Apple mosaic virus and Prunus necrotic ringspot virus.

Apple mosaic virus (ApMV) and Prunus necrotic ringspot virus (PNRSV), belonging to genus Ilarvirus, cause significant losses to rose and other plants of the family Rosaceae. They are easily transmitted through mechanical or vegetative means. In our previous study, the occurrence of ApMV and PNRSV in rose plants was reported. In this study, as a first step towards the development of a colorimetric Reverse Transcriptase - Loop Mediated Isothermal Amplification (RT-LAMP) assay, two primer sets were designed, each containing six primers (F3, B3, FIP, BIP, LF and LB) targeting the coat protein genes of ApMV and PNRSV. After incubation of RT-LAMP reaction mix at an isothermal temperature (65 °C/30 min), the amplified products were visually confirmed with the nucleic acid intercalation dye SYBR Green I and the indicator dye Hydroxy-Naphthol Blue. The developed assays were virus specific and showed no cross amplification. Their sensitivity was 103 times higher than that of the corresponding RT-PCRs. The LAMP assays developed in this study are inexpensive, rapid and reliable for the early detection of ApMV and PNRSV, and could therefore be used in plant quarantine to control the risk of their spread.

A novel array of real-time RT-PCR assays for the rapid pathotyping of type I avian paramyxovirus (APMV-1).

Newcastle disease (ND) caused by virulent avian paramyxovirus type I (APMV-1) is a WOAH and EU listed disease affecting poultry worldwide. ND exhibits different clinical manifestations that may either be neurological, respiratory and/or gastrointestinal, accompanied by high mortality. In contrast, mild or subclinical forms are generally caused by lentogenic APMV-1 and are not subject to notification. The rapid discrimination of virulent and avirulent viruses is paramount to limit the spread of virulent APMV-1. The appropriateness of molecular methods for APMV-1 pathotyping is often hampered by the high genetic variability of these viruses that affects sensitivity and inclusivity. This work presents a new array of real-time RT-PCR (RT-qPCR) assays that enable the identification of virulent and avirulent viruses in dual mode, i.e., through pathotype-specific probes and subsequent Sanger sequencing of the amplification product. Validation was performed according to the WOAH recommendations. Performance indicators on sensitivity, specificity, repeatability and reproducibility yielded favourable results. Reproducibility highlighted the need for assays optimization whenever major changes are made to the procedure. Overall, the new RT-qPCRs showed its ability to detect and pathotype all tested APMV-1 genotypes and its suitability for routine use in clinical samples.

Does Avian Coronavirus Co-Circulate with Avian Paramyxovirus and Avian Influenza Virus in Wild Ducks in Siberia?

Avian coronaviruses (ACoV) have been shown to be highly prevalent in wild bird populations. More work on avian coronavirus detection and diversity estimation is needed for the breeding territories of migrating birds, where the high diversity and high prevalence of Orthomyxoviridae and Paramyxoviridae have already been shown in wild birds. In order to detect ACoV RNA, we conducted PCR diagnostics of cloacal swab samples from birds, which we monitored during avian influenza A virus surveillance activities. Samples from two distant Asian regions of Russia (Sakhalin region and Novosibirsk region) were tested. Amplified fragments of the RNA-dependent RNA-polymerase (RdRp) of positive samples were partially sequenced to determine the species of Coronaviridae represented. The study revealed a high presence of ACoV among wild birds in Russia. Moreover, there was a high presence of birds co-infected with avian coronavirus, avian influenza virus, and avian paramyxovirus. We found one case of triple co-infection in a Northern Pintail (Anas acuta). Phylogenetic analysis revealed the circulation of a Gammacoronavirus species. A Deltacoronavirus species was not detected, which supports the data regarding the low prevalence of deltacoronaviruses among surveyed bird species.

Discovery of Avian Paramyxoviruses APMV-1 and APMV-6 in Shorebirds and Waterfowl in Southern Ukraine.

Emerging RNA virus infections are a growing concern among domestic poultry industries due to the severe impact they can have on flock health and economic livelihoods. Avian paramyxoviruses (APMV; avulaviruses, AaV) are pathogenic, negative-sense RNA viruses that cause serious infections in the respiratory and central nervous systems. APMV was detected in multiple avian species during the 2017 wild bird migration season in Ukraine and studied using PCR, virus isolation, and sequencing. Of 4090 wild bird samples collected, mostly from southern Ukraine, eleven isolates were grown in ovo and identified for APMV serotype by hemagglutinin inhibition test as: APMV-1, APMV-4, APMV-6, and APMV-7. To build One Health's capacity to characterize APMV virulence and analyze the potential risks of spillover to immunologically naïve populations, we sequenced virus genomes in veterinary research labs in Ukraine using a nanopore (MinION) platform. RNA was extracted and amplified using a multiplex tiling primer approach to specifically capture full-length APMV-1 (n = 5) and APMV-6 (n = 2) genomes at high read depth. All APMV-1 and APMV-6 fusion (F) proteins possessed a monobasic cleavage site, suggesting these APMVs were likely low virulence, annually circulating strains. Utilization of this low-cost method will identify gaps in viral evolution and circulation in this understudied but important critical region for Eurasia.

JMM Profile: Avian paramyxovirus type-1 and Newcastle disease: a highly infectious vaccine-preventable viral disease of poultry with low zoonotic potential.

Newcastle disease (ND) is a highly contagious disease of poultry caused by virulent avian paramyxovirus-1 (APMV-1) (previously termed avian avulavirus-1 and avian orthoavulavirus-1). APMV-1 is endemic in poultry in many developing countries, whilst outbreaks still occur in developed countries, affecting both commercial and backyard flocks. ND outbreaks can have substantial economic consequences due to high mortality rates and the imposition of trade restrictions. APMV-1 nucleic acid can be detected from swabs or tissues of suspected cases by PCR. Evidence of infection or vaccination may be demonstrated by the presence of specific antibodies against HN in serum samples. No anti-viral treatments exist, but vaccines are available, although there are currently concerns over their efficacy.

Production of Virus-Free Rose Plants using Meristem-Tip Culture and in vitro Thermotherapy.

<b>Background and Objective:</b> Tissue culture and thermotherapy were proved to be suitable in eliminating viruses of many plants. This study was designed in an attempt to produce virus-free Al-Taif rose plants (<i>Rosa damascena</i> Trigintipetala Dieck) through the practical application of the tissue culture approach and thermotherapy. <b>Materials and Methods:</b> Double Antibody Sandwich-Enzyme-Linked Immunosorbent Assay ( DAS-ELISA) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) techniques were used to detect the presence of <i>Apple mosaic virus</i> (ApMV) and <i>Strawberry latent ringspot virus</i> (SLRV) in rose plant materials collected from Taif, KSA. RT-PCR was more sensitive than DAS-ELISA in detecting the 2 viruses. <b>Results:</b> Three different meristem-tip sterilization methods were compared and results revealed that treatment 3 (T₃: 70% Ethanol for 1.0 min and 15% Clorox (Sodium hypochlorite 5.25%) for 10 min) was the most suitable as 97.78% of cleaned meristem tips survived. Meristem tips with different lengths were thermotherapy-treated for different durations. It was indicated that meristem tips of 0.5 or 1.0 cm and heat-treated at 37^oC for four weeks gave the highest percentage of meristems that were able to differentiate into micro-shoots. <b>Conclusion:</b> RT-PCR detection of ApMV and SLRV revealed that using thermotherapy-treatment, for 4 weeks, of 0.5 cm long meristem tips was successfully applied to eliminate the 2 viruses in 92 and 96% of regenerated plantlets, respectively.

Avian Paramyxovirus 4 Antitumor Activity Leads to Complete Remissions and Long-term Protective Memory in Preclinical Melanoma and Colon Carcinoma Models.

Avulaviruses represent a diverse subfamily of non-segmented negative strand RNA viruses infecting avian species worldwide. To date, 22 different serotypes have been identified in a variety of avian hosts, including wild and domestic birds. APMV-1, also known as Newcastle disease virus (NDV), is the only avulavirus that has been extensively characterized due to its relevance for the poultry industry and, more recently, its inherent oncolytic activity and potential as a cancer therapeutic. An array of both naturally-occurring and recombinant APMV-1 strains has been tested in different preclinical models and clinical trials, highlighting NDV as a promising viral agent for human cancer therapy. To date, the oncolytic potential of other closely related avulaviruses remains unknown. Here, we have examined the in vivo anti-tumor capability of prototype strains of APMV serotypes -2, -3, -4, -6, -7, -8 and -9 in syngeneic murine colon carcinoma and melanoma tumor models. Our studies have identified APMV-4 Duck/Hong Kong/D3/1975 virus as a novel oncolytic agent with greater therapeutic potential than one of the NDV clinical candidate strains, La Sota. Intratumoral administration of the naturally-occurring APMV-4 virus significantly extends survival, promotes complete remission, and confers protection against re-challenge in both murine colon carcinoma and melanoma tumor models. Furthermore, we have designed a plasmid rescue strategy that allows us to develop recombinant APMV-4-based viruses. The infectious clone rAPMV-4 preserves the extraordinary antitumor capacity of its natural counterpart, paving the way to a promising next generation of viral therapeutics.

Detection of Velogenic Avian Paramyxoviruses in Rock Doves in New York City, New York.

Avian paramyxovirus 1 (APMV-1), also known as Newcastle disease virus (NDV), causes severe and economically important disease in poultry around the globe. Although a limited amount of APMV-1 strains in urban areas have been characterized, the role of the urban wild bird population as an APMV-1 reservoir is unclear. Because urban birds may have an important role for long-term circulation of the virus, fecal and swab samples were collected by community scientists from wild birds in New York City (NYC), New York, United States. These samples were screened for APMV-1 and genotypically characterized by sequencing of the complete genome. A total of 885 samples were collected from NYC parks and from a local wildlife rehabilitation clinic from October 2020 through June 2021, and 255 samples obtained from 197 birds have been processed to date. Eight birds (4.1%) screened positive for the APMV-1 nucleoprotein gene by conventional reverse transcription PCR (RT-PCR), and two live viruses were isolated via egg culture. A multibasic F protein cleavage sequence, 112R R K K R F117, an indicator of highly pathogenic velogenic APMV-1 strains, was present in the two samples fully sequenced by next generation sequencing. Phylogenetic analysis of the F gene coding sequence classified both isolates into genotype VI, a diverse and predominant genotype responsible for APMV-1 outbreaks in pigeon and dove species worldwide. IMPORTANCE Here we describe the first large-scale effort to screen for APMV-1 in New York City's wild bird population as part of the New York City Virus Hunters program, a community science initiative. We characterized two isolates of APMV-1, with phylogenetic analyses suggesting diversity in established and circulating strains of pigeon paramyxoviruses. Our isolates are also domestic reference strains for future APMV-1 vaccine developments. Future surveillance in this region may contribute to our understanding of APMV-1's evolution and genetic diversity, as well as inform poultry husbandry and vaccination practices in New York State.

Genetic and evolutionary characterization of avian paramyxovirus type 4 in China.

As an economically important poultry pathogen, avian paramyxovirus serotype 4 (APMV-4) frequently reported and isolated from domestic and wild birds particularly waterfowls worldwide. However, evolutionary dynamics of APMV-4 based on genomic characteristics is lacking. In this study, APMV-4 strain designated JX-G13 was isolated from oropharyngeal and cloacal swab samples of wild birds in China. Phylogenetic analysis revealed APMV-4 strains were divided into four genetic genotypes and China isolates were mainly clustered into Genotype I. The MCMC tree indicated that APMV-4 diverged about 104 years ago with the evolutionary rate of 1.2927 × 10-3 substitutions/site/year. BSP analysis suggested that the effective population size of APMV-4 exhibited a steady state and decreased slowly after 2013. The F gene of APMV-4 was considered relatively conserved among isolates based on nucleotide diversity analysis. Although the F gene was under purifying selection, two positions (5 and 21) located in 3'-UTR were subject to positive selection. Our study firstly presented the evolutionary assessments on the genetic diversity of circulating APMV-4 from wild birds and domestic poultry.

Virophages: association with human diseases and their predicted role as virus killers.

The fascinating discovery of the first giant virus, Acanthamoeba polyphaga mimivirus (APMV), belonging to the family Mimiviridae in 2008, and its associated virophage, Sputnik, have left the world of microbiology awestruck. To date, about 18 virophages have been isolated from different environmental sources. With their unique feature of resisting host cell infection and lysis by giant viruses, analogous to bacteriophage, they have been assigned under the family Lavidaviridae. Genome of T-27, icosahedral-shaped, non-enveloped virophages, consist of dsDNA encoding four proteins, namely, major capsid protein, minor capsid protein, ATPase and cysteine protease, which are essential in the formation and assembly of new virophage particles during replication. A few virophage genomes have been observed to contain additional sequences like PolB, ZnR and S3H. Another interesting characteristic of virophage is that Mimivirus lineage A is immune to infection by the Zamilon virophage through a phenomenon termed MIMIVIRE, resembling the CRISPR-Cas mechanism in bacteria. Based on the fact that giant viruses have been found in clinical samples of hospital-acquired pneumonia and rheumatoid arthritis patients, virophages have opened a novel era in the search for cures of various diseases. This article aims to study the prospective role of virophages in the future of human therapeutics.