Molecular characterization suggests kinetic modulation of expression of accessory viral protein, W, in Newcastle disease virus infected DF1 cells.

Viruses adopt strategies to efficiently utilize their compact genome. Members of the family Paramyxoviridae, exhibit a cotranscriptional RNA editing mechanism wherein polymerase stuttering generates accessory proteins from Phosphoprotein (P) gene. Newcastle disease virus (NDV), an avian paramyxovirus, expresses two accessory proteins, V and W, by RNA editing. While P and V proteins are well studied, very little is known about W protein. Recent studies confirmed W protein expression in NDV and the unique subcellular localization of W proteins of virulent and avirulent NDV. We characterized the W protein of NDV strain Komarov, a moderately virulent vaccine strain. W mRNA expression ranged between 7 and 9% of total P gene transcripts similar to virulent NDV. However, W protein expression, detectable by 6 h, peaked at 24 h and dropped by 48 h post infection in DF1 cells indicating a kinetically regulated expression by the virus. The W protein localized in the nucleus and by mutations, a strong nuclear localization signal was identified in the C-terminal region of W protein. The viral growth kinetics study suggested neither supplementation of W protein nor subcellular localization pattern of the supplemented W protein influenced viral replication in vitro similar to that noticed in avirulent NDV. A cytoplasmic mutant of W protein localized in cytoplasm unlike specific mitochondrial colocalization as recorded in velogenic NDV strain SG10 indicating a possible role of W protein in determining the viral pathogenicity. This study describes for the first time, the distinct features of W protein of moderately virulent NDV. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00813-2.

First report of Duck Hepatitis A virus genotype 2 in India.

Sudden death of ducklings was reported in a duck farm located at Tiruvallur district in Tamil Nadu, India. Disease investigation began with post mortem findings of dead birds revealing enlarged pale-pink / pale-yellow liver with multifocal petechiae and ecchymosis. A positive amplification with duck hepatitis A virus specific primers by reverse transcription-polymerase chain reaction (RT-PCR) on the tissue samples collected from dead birds indicated infection by duck hepatitis A virus (DHAV), an avian picornavirus, known to cause acute and high-mortality in ducklings. The virus isolation was successful in 9-days old embryonated chicken eggs, in primary chicken embryo fibroblast (CEF) cells and from experimentally infected ducklings. The embryonic death on day 5 to 7 post inoculation in chicken embryos with signs of cutaneous hemorrhage, edema and greenish yellow liver together with histopathology of embryonic liver and kidney further confirmed DHAV infection. TEM analysis of the infected allantoic fluid and infected CEF cell culture supernatant showed the presence of spherical shaped, non-enveloped virion particles of ~ 20-38 nm diameter, typical for DHAV. Experimental infection of ducklings with RT-PCR positive tissue supernatant caused 40% to 50% mortality with typical petechial hemorrhages on the surface of liver. Further, histopathological analysis and RT-PCR of the inoculated duckling's tissues confirmed the presence of DHAV. Nucleotide sequencing of the 5'UTR region and VP1 region confirmed duck hepatitis A virus genotype 2 (DHAV-2). To the best of our knowledge, this is the first report of laboratory confirmation of DHAV-2 in India. This study warrants the need for the extensive epidemiological surveillance to understand the prevalence of DHAV-2 in India and to take appropriate control measures to curtail the disease spread.

First complete genome sequence of lumpy skin disease virus directly from a clinical sample in South India.

Lumpy skin disease (LSD), a notifiable disease listed by the World Organization for Animal Health and a fast fast-moving transboundary viral disease infecting cattle and buffaloes, was reported in India in 2019 and has since rapidly spread across the country. This study reports the first complete genome sequence and analysis of a pathogenic LSD virus (LSDV) from India (LSDV/208/PVNRTVU/2020) obtained by direct sequencing of a suspected clinical sample using Illumina and Nanopore sequencing technologies. The complete genome sequence of LSDV/208/PVNRTVU/2020 is 150445 bp long, codes for 156 putative genes and carries identical 2254 bp inverted terminal repeats at either ends. The unique features reported in the LSDV isolates from the recent outbreaks in Asia, namely, the insertions of 12 nucleotides in the viral G-protein coupled receptor (GPCR) and 27 nucleotides leading to duplication of 9 aminoacids in the extracellular enveloped virus-specific (EEV) genes were also conserved in LSDV/208/PVNRTVU/2020. Phylogenetic analysis of the complete genome sequence of LSDV/208/PVNRTVU/2020 revealed its close relation with Kenyan strains and clustered away from vaccine strains. Further analysis showed evidence of strong purifying selection without any recombination events. The data presented in this study could be useful for designing effective strategies such as developing rapid diagnostics and vaccines to control LSD.

Milk exosomes elicit a potent anti-viral activity against dengue virus.

BACKGROUND: Exosomes are nano-sized vesicles secreted by various cells into the intra and extracellular space and hence is an integral part of biological fluids including milk. In the last few decades, many research groups have proved the potential of milk exosomes as a sustainable, economical and non-immunogenic drug delivery and therapeutic agent against different pathological conditions. However, its anti-viral properties still remain to be unearthed. METHODS: Here, we have been able to isolate, purify and characterize the milk derived exosomes from Cow (CME) and Goat (GME) and further studied its antiviral properties against Dengue virus (DENV), Newcastle Disease Virus strain Komarov (NDV-K) and Human Immunodeficiency Virus (HIV-1) using an in-vitro infection system. RESULTS: TEM, NTA and DLS analysis validated the appropriate size of the isolated cow and goat milk exosomes (30-150 nm). Real-time PCR and immunoblotting results confirmed the presence of several milk exosomal miRNAs and protein markers. Our findings suggest that GME significantly decreased the infectivity of DENV. In addition, we confirmed that GME significantly reduces DENV replication and reduced the secretion of mature virions. Furthermore, heat inactivation of GME did not show any inhibition on DENV infection, replication, and secretion of mature virions. RNase treatment of GME abrogates the anti-viral properties indicating direct role of exosomes in DENV inhibition. In addition GME inhibited the infectivity of NDV-K, but not HIV-1, suggesting that the GME mediated antiviral activity might be virus specific. CONCLUSION: This study demonstrates the anti-viral properties of milk exosomes and opens new avenues for the development of exosome-based therapies to treat viral diseases.

Molecular detection and characterization of highly pathogenic porcine reproductive and respiratory syndrome virus from a natural outbreak in wild pigs, Mizoram, India.

This study reports for the first time a natural outbreak of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) caused by HP-PRRS virus (HP-PRRSV) in wild pigs characterized by sudden onset of depression, anorexia, respiratory distress, and high fever. The disease has caused severe haemorrhagic pneumonia, haemorrhagic lymphadenitis, enlarged spleen with areas of infarction, and petechial haemorrhages on the myocardium and on the surface of kidneys. HP-PRRSV was detected in representative tissue samples by reverse transcription-PCR, and the field strain was isolated in the MA104 cell line. The phylogenetic analyses based on the whole genome sequences and nucleotide sequences of open reading frame 5 (ORF5) gene showed close grouping with the subtype IV of lineage 8/8.7 of PRRSV II, which represents the HP-PRRSV strains that predominate in the pig population of China since 2010. The amino acid sequence analysis of the ORF5 gene revealed the replacement of leucine (L) at position 39 to isoleucine (I) in the primary neutralizing epitope. Among the four potential N glycosylation sites, the N34 was mutated and found to be restricted to only three N glycosylation sites. The present findings have indicated that HP-PRRSV can cause fatal outbreaks and may emerge as a major threat to the wild pig population.

Whole genome analysis and molecular characterization of chicken infectious anemia virus from an outbreak in a layer flock reveals circulation of genogroup IIIb in South India.

The complete genome (2298 nucleotides) of the economically important and immunosuppressive, chicken infectious anemia virus (CAV), from a disease outbreak in a layer flock is discussed. This is the first report of a complete genome sequence of CAV from India. The phylogenetic analyses grouped this isolate with CAV genogroup IIIb based on both complete genome and capsid protein (VP1) sequences. The analyses further revealed the presence of CAV genogroups II, IIIa and IIIb in India. The VP1 sequence identity ranged between 84.4 to 99.3% with that of the Indian isolates and carried a unique substitution at position 447 (serine instead of threonine). Two novel amino acid substitutions were observed at position 52 of VP1 (serine instead of proline) and at position 26 of VP2 (asparagine instead of serine). Sequence analyses of VP1, VP2 and VP3 suggested that the isolate could be attenuated. Comparison with CAV variants, isolated from mammalian species, showed similarities in the numbers of certain transcription factor binding sites in the non-coding regions. Recombination analysis detected no recombination events in this isolate. Further investigations are needed to understand the implications of the unique features of this isolate on viral virulence.

Molecular detection and phylogenetic analysis of Marek's disease virus virulence-associated genes from vaccinated flocks in southern India reveals circulation of virulent MDV genotype.

Marek's disease (MD) is a re-emerging viral disease of chickens and a serious economic threat to the poultry industry worldwide. Continuous surveillance with molecular investigation is essential to monitor the emergence of virulent Marek's disease virus (MDV) strains and to devise any appropriate vaccination strategy and implement bio-security programmes. In the present study, we investigated the cases of MD outbreaks in vaccinated poultry flocks. The MD outbreak was confirmed through necropsy (mainly visceral tumours), histopathology and viral gene specific PCR. The pathotypes of the field MDV strains were assessed by molecular analysis of three virulence-associated genes, meq, pp38 and vIL-8. The Meq sequence of the field strains analyzed in this study lacked the 59 aa unique to mild strains, indicating that they are potentially virulent strains. Mutation at position 71 and the presence of five proline rich repeats in the transactivation domain, both associated with virulence were observed in these strains; however, the signature sequences specific to very virulent plus strains were absent. Phylogenetic analysis of meq oncogene sequences revealed clustering of the field strains with North Indian strains and with a very virulent plus ATE 2539 strain from Hungary. Analyses of pp38 protein at positions 107 and 109 and vIL-8 protein at positions 4 and 31 showed signatures of virulence. Sequence and phylogenetic analysis of oncogene and virulence-associated genes of field MDVs from vaccinated flock indicated that these strains possessed molecular features of virulent strains.

Development and validation of high throughput real-time polymerase chain reaction assay for quantitative detection of chicken infectious anemia virus.

A real time polymerase chain reaction (real-time PCR) assay was developed to detect and quantify the chicken infectious anemia virus (CIAV). The two sets of primers specific to VP1 region of CIAV were designed and their sensitivity and efficacy were studied. Both the primers designed in this study were highly sensitive and were able to detect upto 0.01 fg/µl or 82 × 102 copy number of plasmid DNA. The efficiency of the real time PCR was 100.9%. The results have also shown that the present qPCR assay is 100 times more sensitive than regular qualitative PCR. Both primer sets were validated using 28 field poultry samples and showed good results. The optimized real-time quantitative PCR will be useful in quick detection of field outbreaks, sub-clinical infection in poultry flocks, virus pathogenesis studies and for detecting vaccine contamination.

Publisher Correction: Molecular evolution and genetic variations of V and W proteins derived by RNA editing in Avian Paramyxoviruses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular evolution and genetic variations of V and W proteins derived by RNA editing in Avian Paramyxoviruses.

The newly assigned subfamily Avulavirinae in the family Paramyxoviridae includes avian paramyxoviruses (APMVs) isolated from a wide variety of avian species across the globe. Till date, 21 species of APMVs are reported and their complete genome sequences are available in GenBank. The APMV genome comprises of a single stranded, negative sense, non-segmented RNA comprising six transcriptional units (except APMV-6 with seven units) each coding for a structural protein. Additionally, by co-transcriptional RNA editing of phosphoprotein (P) gene, two mRNAs coding for accessory viral proteins, V and W, are generated along with unedited P mRNA. However, in APMV-11, the unedited mRNA codes for V protein while +2 edited mRNA translates to P protein, similar to members of subfamily Rubulavirinae in the same family. Such RNA editing in paramyxoviruses enables maximizing the coding capacity of their smaller genome. The three proteins of P gene: P, V and W, share identical N terminal but varied C terminal sequences that contribute to their unique functions. Here, we analyzed the P gene editing site, V and W sequences of all 21 APMV species known so far (55 viruses) by using bioinformatics and report their genetic variations and molecular evolution. The variations observed in the sequence and hexamer phase positions of the P gene editing sites is likely to influence the levels and relative proportions of P, V and W proteins' expressions which could explain the differences in the pathogenicity of APMVs. The V protein sequences of APMVs had conserved motifs similar to V proteins of other paramyxoviruses including the seven cysteine residues involved in MDA5 interference, STAT1 degradation and interferon antagonism. Conversely, W protein sequences of APMVs were distinct. High sequence homology was observed in both V and W proteins between strains of the same species than between species except in APMV-3 which was the most divergent APMV species. The estimates of synonymous and non-synonymous substitution rates suggested negative selection pressure on the V and W proteins within species indicating their low evolution rate. The molecular clock analysis revealed higher conservation of V protein sequence compared to W protein indicating the important role played by V protein in viral replication, pathogenesis and immune evasion. However, we speculate the genetic diversity of W proteins could impact the degree of pathogenesis, variable interferon antagonistic activity and the wide host range exhibited by APMV species. Phylogenetically, V proteins of APMVs clustered into three groups similar to the recent classification of APMVs into three new genera while no such pattern could be deciphered in the analysis of W proteins except that strains of same species grouped together. This is the first comprehensive study describing in detail the genetic variations and the molecular evolution of P gene edited, accessory viral proteins of Avian paramyxoviruses.

Molecular phylogenetics of Newcastle disease viruses isolated from vaccinated flocks during outbreaks in Southern India reveals circulation of a novel sub-genotype.

Newcastle disease (ND) is an economically important, contagious poultry viral disease reported across the globe. In India, ND is endemic and episodes of ND outbreaks despite strict vaccinations are not uncommon. We isolated and characterized seven ND viruses from vaccinated commercial poultry farms during severe disease outbreaks in Tamil Nadu, in Southern India, between April 2015 and June 2016. All the seven isolates were categorized as virulent by mean death time (48-54 hr) in embryonated chicken eggs. Also, their sequences carried the virulence signature of multi-basic amino acid residues in their fusion protein cleavage site (RRQ/RR/KRF). Phylogenetic and evolutionary distance analyses revealed circulation of a novel sub-genotype of genotype XIII, class II ND viruses, herein proposed as sub-genotype XIIIe. The genetic divergence between the circulating virulent strains and the vaccine strains could possibly explain the disease outbreak in the vaccinated flocks. Further, our study signifies the need to implement routine epidemiological surveillance and to revisit the current vaccination program.

Co-expression of sialic acid receptors compatible with avian and human influenza virus binding in emus (Dromaius novaehollandiae).

Influenza A viruses (IAVs) continue to threaten animal and human health with constant emergence of novel variants. While aquatic birds are a major reservoir of most IAVs, the role of other terrestrial birds in the evolution of IAVs is becoming increasingly evident. Since 2006, several reports of IAV isolations from emus have surfaced and avian influenza infection of emus can lead to the selection of mammalian like PB2-E627K and PB2-D701N mutants. However, the potential of emus to be co-infected with avian and mammalian IAVs is not yet understood. As a first step, we investigated sialic acid (SA) receptor distribution across major organs and body systems of emu and found a widespread co-expression of both SAα2,3Gal and SAα2,6Gal receptors in various tissues that are compatible with avian and human IAV binding. Our results suggest that emus could allow genetic recombination and hence play an important role in the evolution of IAVs.

TLR-4 signalling pathway: MyD88 independent pathway up-regulation in chicken breeds upon LPS treatment.

Toll-like receptors (TLRs) that sense the microbial pathogens are important components of host immune system. TLRs play key roles in the innate defence mechanism against pathogens, in the development of adaptive immunity, and are possibly the major determinants of the susceptibility to infections. To study the resistance pattern in different breeds of chicken, a comprehensive understanding of TLR4 signalling pathways is required. We investigated the TLR-4 pathway regulated gene expressions in PBMCs of chicken breeds of Broiler (Cobb), Aseel, Dahlem Red and Ghagus upon LPS treatment using Quantitative RT-PCR approach. Several genes were found to be up regulated in both TLR-induced MyD88-dependent and MyD88-independent pathways. These genes include TLR4 (Toll-like receptor 4), MyD88 (Myeloid differentiation primary response gene 88), TRAF6 (TNF receptor associated factor 6), TRIF (TIR domain containing adapter inducing interferon beta), the transcription factors NFkB (Nuclear factor kappa B), IRF7 (Interferon regulatory factor 7) and IFN ß (Interferon beta). We have also studied inflammatory cytokines such as IL2, IL6, IL8, IL1 ß and TNF α to further understand the downstream signalling of TLR4 pathway. These results showed that higher expression of TLR signalling activation via both MyD88-dependent and TRIF-dependent pathways are more beneficial to chicken mononuclear cells mediated innate immunity. We observed TRIF dependent pathway in Aseel and Ghagus breeds. Our results are in concurrent with general observation that Aseel breed is comparatively more resistant, Ghagus and broilers are moderately resistant and Dahlem Red is comparatively more susceptible to bacterial infections.

Validation of a human cell based high-throughput genotoxicity assay 'Anthem's Genotoxicity screen' using ECVAM recommended lists of genotoxic and non-genotoxic chemicals.

A novel high throughput-enabled human cell based screen, Anthem's Genotoxicity screen, was developed to achieve higher specificity for predicting in vivo genotoxins by an in vitro method. The assay employs engineered human colon carcinoma cell line; HCT116 cells that are stably engineered with three promoter-reporter cassettes such that an increased reporter activity reflects the activation of associated signaling events in a human cell. The current study focuses on the evaluation of sensitivity and specificity of Anthem's Genotoxicity screen using 62 compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM). The concordance of Anthem's Genotoxicity screen with in vivo tests was 95.5% with sensitivity of 95.2% and specificity of 95.7%. Thus Anthem's Genotoxicity screen, a high-throughput mechanism based genotox indicator test can be employed by a variety of industries for rapid screening and early detection of potential genotoxins.

Mutation of the f-protein cleavage site of avian paramyxovirus type 7 results in furin cleavage, fusion promotion, and increased replication in vitro but not increased replication, tissue tropism, or virulence in chickens.

We constructed a reverse genetics system for avian paramyxovirus serotype 7 (APMV-7) to investigate the role of the fusion F glycoprotein in tissue tropism and virulence. The AMPV-7 F protein has a single basic residue arginine (R) at position -1 in the F cleavage site sequence and also is unusual in having alanine at position +2 (LPSSR↓FA) (underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.). APMV-7 does not form syncytia or plaques in cell culture, but its replication in vitro does not depend on, and is not increased by, added protease. Two mutants were successfully recovered in which the cleavage site was modified to mimic sites that are found in virulent Newcastle disease virus isolates and to contain 4 or 5 basic residues as well as isoleucine in the +2 position: (RRQKR↓FI) or (RRKKR↓FI), named Fcs-4B or Fcs-5B, respectively. In cell culture, one of the mutants, Fcs-5B, formed protease-independent syncytia and grew to 10-fold-higher titers compared to the parent and Fcs-4B viruses. This indicated the importance of the single additional basic residue (K) at position -3. Syncytium formation and virus yield of the Fcs-5B virus was impaired by the furin inhibitor decanoyl-RVKR-CMK, whereas parental APMV-7 was not affected. APMV-7 is avirulent in chickens and is limited in tropism to the upper respiratory tract of 1-day-old and 2-week-old chickens, and these characteristics were unchanged for the two mutant viruses. Thus, the acquisition of furin cleavability by APMV-7 resulted in syncytium formation and increased virus yield in vitro but did not alter virus yield, tropism, or virulence in chickens.

Roles of the fusion and hemagglutinin-neuraminidase proteins in replication, tropism, and pathogenicity of avian paramyxoviruses.

Virulent and moderately virulent strains of Newcastle disease virus (NDV), representing avian paramyxovirus serotype 1 (APMV-1), cause respiratory and neurological disease in chickens and other species of birds. In contrast, APMV-2 is avirulent in chickens. We investigated the role of the fusion (F) and hemagglutinin-neuraminidase (HN) envelope glycoproteins in these contrasting phenotypes by designing chimeric viruses in which the F and HN glycoproteins or their ectodomains were exchanged individually or together between the moderately virulent, neurotropic NDV strain Beaudette C (BC) and the avirulent APMV-2 strain Yucaipa. When we attempted to exchange the complete F and HN glycoproteins individually and together between the two viruses, the only construct that could be recovered was recombinant APMV-2 strain Yucaipa (rAPMV-2), containing the NDV F glycoprotein in place of its own. This substitution of NDV F into APMV-2 was sufficient to confer the neurotropic, neuroinvasive, and neurovirulent phenotypes, in spite of all being at reduced levels compared to what was seen for NDV-BC. When the ectodomains of F and HN were exchanged individually and together, two constructs could be recovered: NDV, containing both the F and HN ectodomains of APMV-2; and APMV-2, containing both ectodomains of NDV. This supported the idea that homologous cytoplasmic tails and matched F and HN ectodomains are important for virus replication. Analysis of these viruses for replication in vitro, syncytium formation, mean embryo death time, intracerebral pathogenicity index, and replication and tropism in 1-day-old chicks and 2-week-old chickens showed that the two contrasting phenotypes of NDV and APMV-2 could largely be transferred between the two backbones by transfer of homotypic F and HN ectodomains. Further analysis provided evidence that the homologous stalk domain of NDV HN is essential for virus replication, while the globular head domain of NDV HN could be replaced with that of APMV-2 with only a minimal attenuating effect. These results demonstrate that the F and HN ectodomains together determine the cell fusion, tropism, and virulence phenotypes of NDV and APMV-2 and that the regions of HN that are critical to replication and the species-specific phenotypes include the cytoplasmic tail and stalk domain but not the globular head domain.

Mutations in the fusion protein cleavage site of avian paramyxovirus serotype 2 increase cleavability and syncytium formation but do not increase viral virulence in chickens.

Avian paramyxovirus serotype 2 (APMV-2) is one of the nine serotypes of APMV, which infect a wide variety of avian species around the world. In this study, we constructed a reverse genetics system for recovery of infectious recombinant APMV-2 strain Yucaipa (APMV-2/Yuc) from cloned cDNA. The rescued recombinant virus (rAPMV-2) resembled the biological virus in growth properties in vitro and in pathogenicity in vivo. The reverse genetics system was used to analyze the role of the cleavage site of the fusion (F) protein in viral replication and pathogenesis. The cleavage site of APMV-2/Yuc (KPASR↓F) contains only a single basic residue (position -1) that matches the preferred furin cleavage site [RX(K/R)R↓]. (Underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.) Contrary to what would be expected for this cleavage sequence, APMV-2 does not require, and is not augmented by, exogenous protease supplementation for growth in cell culture. However, it does not form syncytia, and the virus is avirulent in chickens. A total of 12 APMV-2 mutants with F protein cleavage site sequences derived from APMV serotypes 1 to 9 were generated. These sites contain from 1 to 5 basic residues. Whereas a number of these cleavage sites are associated with protease dependence and lack of syncytium formation in their respective native viruses, when transferred into the APMV-2 backbone, all of them conferred protease independence, syncytium formation, and increased replication in cell culture. Examination of selected mutants during a pulse-chase experiment demonstrated an increase in F protein cleavage compared to that for wild-type APMV-2. Despite the gains in cleavability, replication, and syncytium formation, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old chicks, and 2-week-old chickens showed that the F protein cleavage site mutants did not exhibit increased pathogenicity and remained avirulent. These results imply that structural features in addition to the cleavage site play a major role in the cleavability of the F protein and the activity of the cleaved protein. Furthermore, cleavage of the F protein is not a determinant of APMV-2 pathogenicity in chickens.

Experimental infection of hamsters with avian paramyxovirus serotypes 1 to 9.

Avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world and are separated into nine serotypes (APMV-1 to -9). Only in the case of APMV-1, the infection of non-avian species has been investigated. The APMVs presently are being considered as human vaccine vectors. In this study, we evaluated the replication and pathogenicity of all nine APMV serotypes in hamsters. The hamsters were inoculated intranasally with each virus and monitored for clinical disease, pathology, histopathology, virus replication, and seroconversion. On the basis of one or more of these criteria, each of the APMV serotypes was found to replicate in hamsters. The APMVs produced mild or inapparent clinical signs in hamsters except for APMV-9, which produced moderate disease. Gross lesions were observed over the pulmonary surface of hamsters infected with APMV-2 & -3, which showed petechial and ecchymotic hemorrhages, respectively. Replication of all of the APMVs except APMV-5 was confirmed in the nasal turbinates and lungs, indicating a tropism for the respiratory tract. Histologically, the infection resulted in lung lesions consistent with bronchointerstitial pneumonia of varying severity and nasal turbinates with blunting or loss of cilia of the epithelium lining the nasal septa. The majority of APMV-infected hamsters exhibited transient histological lesions that self resolved by 14 days post infection (dpi). All of the hamsters infected with the APMVs produced serotype-specific HI or neutralizing antibodies, confirming virus replication. Taken together, these results demonstrate that all nine known APMV serotypes are capable of replicating in hamsters with minimal disease and pathology.

Pathogenesis of two strains of avian paramyxovirus serotype 2, Yucaipa and Bangor, in chickens and turkeys.

Nine serologic types of avian paramyxovirus (APMV) have been recognized. Newcastle disease virus (APMV-1) is the most extensively characterized virus, while relatively little information is available for the other APMV serotypes. In the present study, we examined the pathogenicity of two strains of APMV-2, Yucaipa and Bangor, in 9-day-old embryonated chicken eggs, 1-day-old specific-pathogen-free (SPF) chicks, and 4-wk-old SPF chickens and turkeys. The mean death time in 9-day-old embryonated chicken eggs was more than 168 hr for both strains, and their intracerebral pathogenicity index (ICPI) was zero, indicating that these viruses are nonpathogenic in chickens. When inoculated intracerebrally in 1-day-old chicks, neither strain caused disease or replicated detectably in the brain. This suggests that the zero ICPI value of APMV-2 reflects the inability of the virus to grow in neural cells. Groups of twelve 4-wk-old SPF chickens and turkeys were inoculated oculonasally with either strain, and three birds per group were euthanatized on days 2, 4, 6, and 14 postinoculation for analysis. There were no overt clinical signs of illnesses, although all birds seroconverted by day 6. The viruses were isolated predominantly from the respiratory and alimentary tracts. Immunohistochemistry studies also showed the presence of a large amount of viral antigens in epithelial linings of respiratory and alimentary tracts. There also was evidence of systemic spread even though the cleavage site of the viral fusion glycoprotein does not contain the canonical furin protease cleavage site.

Complete genome sequences of avian paramyxovirus serotype 2 (APMV-2) strains Bangor, England and Kenya: evidence for the existence of subgroups within serotype 2.

The complete consensus genome sequences of avian paramyxovirus (APMV) serotype 2 strains Bangor, England and Kenya were determined and compared with those of APMV-2 prototype strain Yucaipa and other paramyxoviruses. The genome lengths of APMV-2 strains Bangor, England and Kenya are 15,024, 14,904, 14,916 nucleotides (nt), respectively, compared to 14,904 nt for Yucaipa. Each genome consists of six non-overlapping genes in the order of (3)(')N-P/V/W-M-F-HN-L(5'), with a 55-nt leader at the 3' end. The length of the trailer at the 5' end of strain Bangor was 173 nt, compared to 154 nt for strains England, Kenya, and Yucaipa. In general, sequence comparison of APMV-2 strains England and Kenya with strain Yucaipa have 94.5 and 88% nt and 96 and 92% aggregate amino acid (aa) identity, respectively. In contrast, strain Bangor has a much lower percent nt identity (70.4, 69.4, and 70.8%) and aa identity (75.3, 76.2, and 76.3%) with strains Yucaipa, England, and Kenya, respectively. Furthermore, strain Bangor has a single basic aa residue ((101)TLPSAR F(108)) at the fusion protein cleavage site compared to the dibasic aa ((93)DKPASR F(100)) found in those of other three strains. Reciprocal cross-hemagglutination inhibition (HI) and cross-neutralization assays using post-infection chicken sera indicated that strain Bangor is antigenically related to the other APMV-2 strains, but with a 4- to 8-fold difference in homologous versus heterologous HI titer. These differences in antigenic relatedness suggest that these four APMV-2 strains represent a single serotype with two antigenic subgroups, and this is strongly supported by the dimorphism in nt and aa sequence identity.