Comparison of Crimean-Congo Hemorrhagic Fever Virus and Aigai Virus in Life Cycle Modeling Systems Reveals a Difference in L Protein Activity.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that causes a severe, often fatal, hemorrhagic disease throughout Africa, Asia, and Southeast Europe. A wide variety of strains are circulating in the field which broadly correlate to their geographic distribution. The viral determinants of pathogenicity remain unclear, as does the contribution of strain-specific differences to pathology. Aigai virus (AIGV) is a closely related virus (formally designated CCHFV genotype VI, Europe II, or AP92-like virus), which has been proposed to be less virulent than CCHFV. However, the molecular details leading to potential differences in virulence are unknown. To explore if differences exist, life cycle modeling systems, including both a minigenome and a transcriptionally competent virus-like particle assay, were developed for AIGV to allow the comparison with the CCHFV reference IbAr10200 strain. Using this approach, we could demonstrate that AIGV exhibits lower viral gene expression than the reference strain of CCHFV. Subsequent systematic exchange of viral components revealed that the L protein is responsible for the observed differences in gene expression and that the interferon (IFN) antagonistic activity of the ovarian tumor-type protease domain is not responsible for this effect. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is the cause of severe hemorrhagic disease, which is often fatal. Present throughout Africa, Asia, and Southeast Europe, a diverse number of viral genotypes exist. However, the viral determinants of pathogenicity remain unclear. It has been proposed that the closely related Aigai virus (AIGV) may be a less virulent virus. Here, using newly developed and improved life cycle modeling systems we have examined potential differences between the CCHFV reference strain, IbAr10200, and AIGV. Using this approach, we identified lower viral gene expression driven by the AIGV viral polymerase as a major difference which may be indicative of lower virulence.

A comparative evaluation of transcriptome changes in lung and spleen tissues of chickens infected with velogenic and mesogenic Avian Orthoavulavirus 1.

Newcastle disease is an acute, highly contagious disease responsible for severe economic losses to the poultry industry worldwide. Clinical assessment of different pathotypes of AOaV-1 strains is well-elucidated in chickens. However, a paucity of data exists for a comparative assessment of avian innate immune responses in birds after infection with two different pathotypes of AOaV-1. We compared early immune responses in chickens infected with a duck-originated velogenic strain (high virulent: genotype VII) and a pigeon-originated mesogenic stain (moderate virulent; genotype VI). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) identified 4737 differentially expressed genes (DEGs) in the transcriptional profiles of lung and spleen tissues of chickens infected with both pathotypes. More DEGs were expressed in spleen tissue infected with velogenic strain compared to spleen or lung exposed to mesogenic strain. An enriched expression was observed for genes involved in metabolic processes and cellular components, including innate immune-associated signaling pathways. Most DEGs were involved in RIG-I, Toll-like, NF-Kappa B, and MAPK signaling pathways to activate interferon-stimulated genes (ISGs). This study provided a comparative insight into complicated molecular mechanisms and associated DEGs involved in early immune responses of birds to two different AOaV-1 strains.

Phylogenetic assessment reveals continuous evolution and circulation of pigeon-derived virulent avian avulaviruses 1 in Eastern Europe, Asia, and Africa.

BACKGROUND: The remarkable diversity and mobility of Newcastle disease viruses (NDV) includes virulent viruses of genotype VI. These viruses are often referred to as pigeon paramyxoviruses 1 because they are normally isolated and cause clinical disease in birds from the Columbidae family. Genotype VI viruses occasionally infect, and may also cause clinical disease in poultry. Thus, the evolution, current spread and detection of these viruses are relevant to avian health. RESULTS: Here, we describe the isolation and genomic characterization of six Egyptian (2015), four Pakistani (2015), and two Ukrainian (2007, 2013) recent pigeon-derived NDV isolates of sub-genotype VIg. These viruses are closely related to isolates from Kazakhstan, Nigeria and Russia. In addition, eight genetically related NDV isolates from Pakistan (2014-2016) that define a new sub-genotype (VIm) are described. All of these viruses, and the ancestral Bulgarian (n = 2) and South Korean (n = 2) viruses described here, have predicted virulent cleavage sites of the fusion protein, and those selected for further characterization have intracerebral pathogenicity index assay values characteristic of NDV of genotype VI (1.31 to 1.48). A validated matrix gene real-time RT-PCR (rRT-PCR) NDV test detect all tested isolates. However, the validated rRT-PCR test that is normally used to identify the virulent fusion gene fails to detect the Egyptian and Ukrainian viruses due to mismatches in primers and probe. A new rapid rRT-PCR test to determine the presence of virulent cleavage sites for viruses from sub-genotypes VIg was developed and evaluated on these and other viruses. CONCLUSIONS: We describe the almost simultaneous circulation and continuous evolution of genotype VI Newcastle disease viruses in distant locations, suggesting epidemiological connections among three continents. As pigeons are not migratory, this study suggests the need to understand the possible role of human activity in the dispersal of these viruses. Complete genomic characterization identified previously unrecognized genetic diversity that contributes to diagnostic failure and will facilitate future evolutionary studies. These results highlight the importance of conducting active surveillance on pigeons worldwide and the need to update existent rapid diagnostic protocols to detect emerging viral variants and help manage the disease in affected regions.

Cryptosporidium galli and novel Cryptosporidium avian genotype VI in North American red-winged blackbirds (Agelaius phoeniceus).

Proventriculus and intestinal samples from 70 North American red-winged blackbirds (Agelaius phoeniceus; order Passeriformes) were examined for the presence of Cryptosporidium by PCR amplification and sequence analysis of the 18S ribosomal RNA (18S rRNA), actin, and 70-kDa heat shock protein (HSP70) genes. Twelve birds (17.1 %) were positive for the Cryptosporidium 18S rRNA gene: six birds were positive at the proventriculus site only and six birds were positive at the proventriculus and intestinal sites. Sequence analysis of the 18S rRNA, actin and HSP70 genes showed the presence of the gastric species Cryptosporidium galli in a single proventriculus sample and a closely related genotype, which we have named Cryptosporidium avian genotype VI, in all other positive samples. These findings contribute to our understanding of Cryptosporidium diversification in passerines, the largest avian order.

Genetic Characterization, Pathogenicity, and Epidemiology Analysis of Three Sub-Genotype Pigeon Newcastle Disease Virus Strains in China.

Pigeon Newcastle disease (ND) is a serious infectious illness caused by the pigeon Newcastle disease virus (NDV) or Paramyxovirus type 1 (PPMV-1). Genotype VI NDV is a primary factor in ND among Columbiformes (such as pigeons and doves). In a recent study, eight pigeon NDV strains were discovered in various provinces in China. These viruses exhibited mesogenic characteristics based on their MDT and ICPI values. The complete genome sequences of these eight strains showed a 90.40% to 99.19% identity match with reference strains of genotype VI, and a 77.86% to 80.45% identity match with the genotype II vaccine strain. Additionally, analysis of the F gene sequence revealed that these NDV strains were closely associated with sub-genotypes VI.2.2.2, VI.2.1.1.2.1, and VI.2.1.1.2.2. The amino acid sequence at the cleavage site of the F protein indicated virulent characteristics, with the sequences 112KRQKRF117 and 112RRQKRF117 observed. Pigeons infected with these sub-genotype strains had a low survival rate of only 20% to 30%, along with lesions in multiple tissues, highlighting the strong spread and high pathogenicity of these pigeon NDV strains. Molecular epidemiology data from the GenBank database revealed that sub-genotype VI.2.1.1.2.2 strains have been prevalent since 2011. In summary, the findings demonstrate that the prevalence of genotype VI NDV is due to strains from diverse sub-genotypes, with the sub-genotype VI.2.1.1.2.2 strain emerging as the current epidemic strain, highlighting the significance of monitoring pigeon NDV in China.

Detection of Avian Orthoavulavirus-1 genotypes VI.2.1 and VII.1.1 with neuro-viscerotropic tropism in some backyard pigeons (Columbidae) in Eastern Saudi Arabia.

Introduction: Avian orthoavulavirus-1 (AOAV1) has a wide host range, including domestic and wild birds. The present study aimed to identify the currently circulating AOAV1 strains from some outbreaks in some backyard pigeons in the eastern region of Saudi Arabia (ERSA). Methods: Tracheal/cloacal swabs and tissue specimens were collected from eight backyards in Al-Ahsa, ERSA, between January 2021 and March 2023. Samples were tested for the presence of AOAV1 using commercial real-time RT-PCR. Part of the fusion gene was also amplified by gel-based RT-PCR, and the obtained amplicons were sequenced. Results and discussion: AOAV1 was detected in samples from the eight flocks. The retrieved sequences from samples of 6/8 pigeon backyards are reported. Phylogenetic analysis based on the obtained sequences from these backyard pigeons showed the segregation of the obtained sequences in AOAV1 genotypes VI.2.1 and VII.1.1. Clinically, nervous manifestations were dominant in pigeons infected with both genotypes. Respiratory manifestations and significantly higher overall mortality rate were induced by genotype VI.2.1. The deduced amino acid sequences of the fusion protein cleavage site (FPCS) showed that all the detected isolates belong to velogenic strains. Differences in clinical profiles induced by the natural infection of pigeons with AOAV1 genotypes VI.2.1 and VII.1.1 were reported. The present findings highlight the potential roles of some backyard pigeons in the long-distance spread and cross-species transmission of the reported AOAVI genotypes. Further research is required to perform biotyping and pathotyping of the reported strains.

Phylogenetic analysis and pathogenicity assessment of pigeon paramyxovirus type 1 circulating in China during 2007-2019.

Pigeon paramyxovirus type 1 (PPMV-1) is an antigenic variant of Newcastle disease virus (NDV) which is mainly associated with infections of pigeons and has the potential to result in disease in chickens. In this study, we characterised 21 PPMV-1 isolates from diseased pigeons in China during 2007-2019. Phylogenetic analysis revealed that all isolates belonged to genotype VI. Among them, most isolates belonged to sub-genotype VI.2.1.1.2.2, suggesting that VI.2.1.1.2.2 has become a prevalent genotype in pigeons in China. The results showed that all PPMV-1 isolates were mesogenic in nature according to the mean death time (MDT) and intracerebral pathogenicity index (ICPI). In vitro and in vivo studies demonstrated that two genetically closely related isolates (Pi-11 and Pi-10) both of which belonged to sub-genotype VI.2.1.1.2.2 had similar replication kinetics in cells derived from pigeons, while the replication titre of Pi-11 was significantly higher than that of Pi-10 in cells derived from chickens. Pi-11 and Pi-10 could contribute to morbidity and mortality in pigeons. Remarkably, although the two viruses resulted in no apparent disease symptom in chickens, Pi-11 could cause more severe histopathological lesions and had a stronger replication ability in chickens compared to Pi-10. Moreover, chickens infected with Pi-11 had higher shedding efficiency than chickens infected with Pi-10. Additionally, several mutations within important functional regions of the fusion (F) and haemagglutinin-neuraminidase (HN) proteins might be associated with different pathogenicity of the two viruses in chickens. Collectively, these results indicated that the Pi-11-like virus of pigeon origin has the potential to induce severe outbreaks in chicken flocks. These findings will help us better understand the epidemiology and evolution of PPMV-1 in China and serve as a foundation for the further investigation of the mechanism underlying the pathogenic difference of PPMV-1 isolates in chickens.

A molecular, epidemiological and pathogenicity analysis of pigeon paramyxovirus type 1 viruses isolated from live bird markets in China in 2014-2021.

To expand our understanding of the epidemiology of pigeon paramyxovirus type 1 (PPMV-1) in China, risk-based active surveillance was undertaken with pigeon swabs collected from live bird markets in 2014-2021. Seventy-six PPMV-1 strains were isolated from 12 provinces (60%) of the 20 provinces surveyed, and the positive rates of PPMV-1 varied from 0.50% to 3.19% annually. The complete genomic sequences of 18 representative viruses were analyzed, revealing a genome of 15,192 nucleotides, with the gene order 3'-NP-P-M-F-HN-L-5'. All isolates contained the 112RRQKRF117 cleavage site in the fusion (F) protein, a characteristic generally associated with virulent Newcastle disease viruses (NDVs), and the intracerebral pathogenicity index values (1.05-1.41) of four isolates indicated their virulence. A challenge experiment also demonstrated that all four isolates are pathogenic to pigeons, with morbidity rates of 60-100% and mortality rates of 0-30%. A further analysis of the functional domains of the F and HN proteins revealed several mutations in the fusion peptide, signal peptide, neutralizing epitopes, heptad repeat region, and transmembrane domains, and the substitution of cysteine residue 25 (C25Y) and substitutions in the HRb region (V287I) of the F protein and the transmembrane domain (V45A) of the HN protein may play important roles in PPMV-1 virulence. In a phylogenetic analysis based on the complete sequences of the F gene, all eighteen isolates all clustered into sub-genotype VI.2.1.1.2.2 (VIb) in class II, and shared high nucleotide sequence identity, indicating that the PPMV-1 strains in sub-genotype VI.2.1.1.2.2 are the predominant PPMV-1 viruses in pigeons in China and that the variations in these viruses have been relatively stable over the past 8 years. This study identifies the genetic and pathogenicity characteristics of the PPMV-1 strains prevalent in China and extends our understanding of the prevalence of this virus in China.

Evolutionary Dynamics and Age-Dependent Pathogenesis of Sub-Genotype VI.2.1.1.2.2 PPMV-1 in Pigeons.

Pigeon paramyxovirus type 1 (PPMV-1) infection causes high morbidity in pigeons, resulting in a significant burden to the poultry industry. In this study, we isolated three PPMV-1 strains from diseased pigeons collected in Guangdong Province, South China, from June 2017 to April 2019. Genetic analysis revealed that these three PPMV-1 strains and most of the PPMV-1 strains isolated from China after 2011 were clustered into sub-genotype VI.2.1.1.2.2. Our Bayesian analysis revealed that the VI.2.1.1.2.2 viruses might have originated in Europe. Phylogeographic analyses revealed that East and South China might have played a key role in seeding the VI.2.1.1.2.2 PPMV-1 epidemic in China. To characterize the effect of age at infection on the outcome of PPMV-1 infection in pigeons, we investigated the pathogenesis and transmission of the pigeon/Guangdong/GZ08/2017 (GZ08) virus in 3-, 6-, and 12-week-old pigeons. Two of six 12-week-old pigeons inoculated with GZ08 survived, and all of the 3- and 6-week-pigeons inoculated with GZ08 died. Moreover, the GZ08 virus could be transmitted to 3-, 6-, and 12-week-old naïve contact pigeons. The lethality of the GZ08 virus through contact with 3-, 6-, and 12-week-old pigeons was 100%, 66.7%, and 0%, respectively, suggesting that the transmissibility of the GZ08 virus was stronger in young pigeons. These findings demonstrated that East and South China was the epicenter for dissemination of VI.2.1.1.2.2 PPMV-1, and age at infection has an impact on the outcome of PPMV-1 infection in pigeons.