Isolation, characterization, and circulation sphere of a filovirus in fruit bats.

Bats are associated with the circulation of most mammalian filoviruses (FiVs), with pathogenic ones frequently causing deadly hemorrhagic fevers in Africa. Divergent FiVs have been uncovered in Chinese bats, raising concerns about their threat to public health. Here, we describe a long-term surveillance to track bat FiVs at orchards, eventually resulting in the identification and isolation of a FiV, Dehong virus (DEHV), from Rousettus leschenaultii bats. DEHV has a typical filovirus-like morphology with a wide spectrum of cell tropism. Its entry into cells depends on the engagement of Niemann-Pick C1, and its replication is inhibited by remdesivir. DEHV has the largest genome size of filoviruses, with phylogenetic analysis placing it between the genera Dianlovirus and Orthomarburgvirus, suggesting its classification as the prototype of a new genus within the family Filoviridae. The continuous detection of viral RNA in the serological survey, together with the wide host distribution, has revealed that the region covering southern Yunnan, China, and bordering areas is a natural circulation sphere for bat FiVs. These emphasize the need for a better understanding of the pathogenicity and potential risk of FiVs in the region.

First report of haemosporidia and associated risk factors in red junglefowl (Gallus gallus) in China.

BACKGROUND: Avian haemosporidia infect both domestic and wild birds, causing anemia, acute tissue degeneration, and depopulation in wild birds. Poultry and wild birds have been reported as common reservoirs of haemosporidia, but limited information is available for red junglefowl (Gallus gallus) in China. The present study investigated the prevalence and molecular characterization of haemosporidia in red junglefowl. METHODS: Blood samples were collected from 234 red junglefowl from Jinghong City of Yunnan Province, and genomic DNA was extracted from these samples. The prevalence of haemosporidia was determined by nested PCR targeting the mitochondrial cytochrome b (cytb) gene. Molecular characterization was investigated based on phylogenetic analysis of cytb sequences, and associated risk factors were analyzed using the Chi-square (χ2) test. RESULTS: The overall prevalence of haemosporidia was 74.8% (175/234), and three species were identified, namely Haemoproteus enucleator, Leucocytozoon californicus, and Plasmodium juxtanucleare. The prevalence of haemosporidia in adult fowl (81.1%, 107/132) was significantly higher (χ2 = 6.32, df = 1, P = 0.012) than that in juveniles (66.7%, 68/102). Three novel haemosporidian lineages were revealed. CONCLUSIONS: This study examined the prevalence and identified species of avian haemosporidians in red junglefowl, providing new information on the molecular epidemiology and geographical distribution of haemosporidian parasites. Our results indicated high prevalence and diverse species distribution of these haemosporidians in red junglefowl. To the best of our knowledge, this is the first record of haemosporidian infection in red junglefowl in China.

Clade 2.3.4.4b H5N8 Subtype Avian Influenza Viruses Were Identified from the Common Crane Wintering in Yunnan Province, China.

The seasonal migration of wild aquatic birds plays a critical role in the maintenance, transmission, and incursion of the avian influenza virus (AIV). AIV surveillance was performed during 2020-2021 in two national nature reserves with abundant wild bird resources in Yunnan, China. Four H5N8 AIVs isolates from the common crane were identified by next-generation sequencing. Phylogenetic analysis demonstrated that all eight gene segments of these H5N8 AIVs belonged to clade 2.3.4.4b high-pathogenic AIV (HPAIV) and shared high nucleotide sequence similarity with the strains isolated in Hubei, China, and Siberia, Russia, in 2020-2021. The H5N8 HPAIVs from common cranes were characterized by both human and avian dual-receptor specificity in the hemagglutinin (HA) protein. Moreover, possessing the substitutions contributes to overcoming transmission barriers of mammalian hosts in polymerase basic 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acid (PA), and exhibiting the long stalk in the neck region of the neuraminidase (NA) protein contributes to adaptation in wild birds. Monitoring AIVs in migratory birds, at stopover sites and in their primary habitats, i.e., breeding or wintering grounds, could provide insight into potential zoonosis caused by AIVs.

Emergence of novel clade 2.3.4 influenza A (H5N1) virus subgroups in Yunnan Province, China.

From December 2013 to March 2014, a major wave of highly pathogenic avian influenza outbreak occurred in poultry in Yunnan Province, China. We isolated and characterized eight highly pathogenic avian influenza A (H5N1) viruses from poultry. Full genome influenza sequences and analyses have been performed. Sequence analyses revealed that they belonged to clade 2.3.4 but did not fit within the three defined subclades. The isolated viruses were provisional subclade 2.3.4.4e. The provisional subclade 2.3.4.4e viruses with six internal genes from avian influenza A (H5N2) viruses in 2013 were the novel reassortant influenza A (H5N1) viruses which were associated with the outbreak of H5N1 occurred in egg chicken farms in Yunnan Province. The HA genes were similar to subtype H5 viruses isolated from January to March of 2014 in Asia including H5N6 and H5N8. The NA genes were most closely related to A/chicken/Vietnam/NCVD-KA423/2013 (H5N1) from the subclade 2.3.2. The HI assay demonstrated a lack of antigenic relatedness between clades 2.3.4.4e and 2.3.4.1 (RE-5 vaccine strain) or 2.3.2.2 (RE-6 vaccine strain).

[Genetic evolution analysis of matrix protein 2 gene of avian influenza H5N1 viruses from boundary of Yunnan province].

OBJECTIVE: To elucidate the variation in characterizations and genetic evolution of the matrix protein 2 or ion channel protein(M2) genes of avian influenza subtype H5N1 viruses in the boundary region of Yunnan province from 2008 to 2012. METHODS: A total of swab samples were collected from foreign poultry such as the junction between Yunnan and Vietnam, Laos,myanmar and wild birds in boundary region of Yunnan province from 2008 to 2012 and screened by H5N1 subtype-specific multiplex RT-PCR. The M genes of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. The alignment and phylogenetic analysis of M2 genes were performed with sequences of the known reference strains. RESULTS: A total of 71 positive samples were found out of 1240 samples and the positive rate was 5.72%. A total of 14 different M2 sequences were obtained from 30 positive samples and were divided into 3 distinct clades or sub-clades(1.2.1, 1.2.2 and 2) by phylogenetic analysis, 5, 7 and 2, respectively. The M2 genes and Hemagglutinin(HA) genes of H5N1 viruses from the boundary region of Yunnan province had showed different relationship of genetic evolution. The substitution or mutation of key amino acids sites had been found among the domains of epitope, adamantane-resistance, and poultry or human original viral strains. CONCLUSION: The M2 genes of H5N1 subtype viruses in boundary region of Yunnan province from 2008 to 2012 showed genetic divergence and the virus of clade 1.2.2 had become dominant epidemic strain in this region.

[Genetic evolution of non-structural gene among avian influenza H5N1 viruses isolated from the boundary of Yunnan province].

OBJECTIVE: To elucidate the characteristics of variation and the genetic evolution of non-structural protein (NS1, NS2) genes related to avian influenza subtype H5N1 viruses isolated from the boundary region of Yunnan province. METHODS: Swab samples were collected from foreign poultry and wild birds in the boundary regions of Yunnan province and screened by H5/N1 subtype-specific multiplex RT-PCR. The NS segment of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. The alignment and phylogenetic analysis on those available NS1, NS2 genes were performed with sequences of the known reference strains. RESULTS: 71 positive samples were identified from 1240 samples, with the positive rate as 5.72%. Fourteen different NS segment sequences were obtained from 30 representative positive samples and could be divided into 3 distinct clades or sub-clades (I-1, I-2 and II), by phylogenetic analysis. The NS1/NS2 genes and Hemagglutinin (HA) genes of H5N1 viruses from the boundary regions of Yunnan province showed different relationships regarding the characteristics on genetic evolution. The substitution or mutation of key amino acids sites had been noticed in the nuclear location signal domains, effect domain, and other pathogenicity markers. CONCLUSION: NS genes of H5N1 subtype viruses in boundary region of Yunnan province showed genetic divergence and the virus of clade I-2 and II had become dominant epidemic strains in this region since 2010.