H6N2 reassortant avian influenza virus isolate in wild birds in Jiangxi Province, China.

H6 avian influenza virus is widely prevalent in wild birds and poultry and has caused human infection in 2013 in Taiwan, China. During our active influenza surveillance program in wild waterfowl at Poyang Lake, Jiangxi Province, an H6N2 AIV was isolated and named A/bean goose/JiangXi/452-4/2013(H6N2). The isolate was characterized as a typical low pathogenic avian influenza virus (LPAIV) due to the presence of the amino acid sequence PQIETR↓GLFGAI at the cleavage site of the hemagglutinin (HA) protein. The genetic evolution analysis revealed that the NA gene of the isolate originated from North America and exhibited the highest nucleotide identity (99.29%) with a virus recovered from wild bird samples in North America, specifically A/bufflehead/California/4935/2012(H11N2). Additionally, while the HA and PB1 genes belonged to the Eurasian lineage, they displayed frequent genetic interactions with the North American lineage. The remaining genes showed close genetic relationships with Eurasian viruses. The H6N2 isolate possessed a complex genome, indicating it is a multi-gene recombinant virus with genetic material from both Eurasian and North American lineages.

Emergence, Evolution, and Pathogenicity of Influenza A(H7N4) Virus in Shorebirds in China.

A 2-year surveillance study of influenza A viruses in migratory birds was conducted to understand the subsequent risk during the migratory seasons in Dandong Yalu River Estuary Coastal Wetland National Nature Reserve, Liaoning Province, China, a major stopover site on the East Asian-Australasian flyway. Overall, we isolated 27 influenza A viruses with multiple subtypes, including H3N8 (n = 2), H4N6 (n = 2), H4N7 (n = 2), H7N4 (n = 9), H7N7 (n = 1), H10N7 (n = 7), and H13N6 (n = 4). Particularly, a novel reassortant influenza A(H7N4) virus was first identified in a woman and her backyard poultry flock in Jiangsu Province, China, posing a serious threat to public health. Here, we describe the genetic characterization and pathogenicity of the nine influenza A(H7N4) isolates. Phylogenetic analysis indicated that complex viral gene flow occurred among Asian countries. We also demonstrated a similar evolutionary trajectory of the surface genes of the A(H7N4) isolates and Jiangsu human-related A(H7N4) viruses. Our A(H7N4) isolates exhibited differing degrees of virulence in mice, suggesting a potential risk to other mammalian species, including humans. We revealed multiple mutations that might affect viral virulence in mice. Our report highlights the importance and need for the long-term surveillance of avian influenza virus in migratory birds combined with domestic poultry surveillance along migratory routes and flyways and, thereby, the development of measures to manage potential health threats. IMPORTANCE The H7 subtype avian influenza viruses, such as H7N2, H7N3, H7N4, H7N7, and H7N9, were documented as being capable of infecting humans, and the H7 subtype low pathogenicity avian influenza viruses are capable of mutating into highly pathogenic avian influenza; therefore, they pose a serious threat to public health. Here, we investigated the evolutionary history, molecular characteristics, and pathogenicity of shorebird-origin influenza A(H7N4) viruses, showing a similar evolutionary trajectory with Jiangsu human A(H7N4) viruses in HA and NA genes. Moreover, our isolates exhibited variable virulence (including moderate virulence) in mice, suggesting a potential risk to other mammalian species, including humans.

Seroprevalence and risk factor analysis of Toxoplasma gondii infection in Siberian tigers (Panthera tigris altaica) and giant pandas (Ailuropoda melanoleuca) in China.

Toxoplasmosis, caused by Toxoplasma gondii, is a worldwide zoonosis. The aim of the present study was to detect the seroprevalence of T. gondii infection and associated risk factors among Siberian tigers (Panthera tigris altaica) and giant pandas (Ailuropoda melanoleuca) in China. Blood samples from 112 Siberian tigers and 22 giant pandas were tested for immunoglobulin G (IgG) against T. gondii by enzyme-linked immunosorbent assay (ELISA). The seroprevalence of T. gondii infection was 7.14% among Siberian tigers and 9.09% among giant pandas. No risk factors were found to be significantly associated with seroprevalence (P > 0.05). This is the first study to evaluate T. gondii infection in Siberian tigers on a large scale in China, and it also updates the information regarding the positivity rate of T. gondii infection among giant pandas in China.

Equus roundworms (Parascaris univalens) are undergoing rapid divergence while genes involved in metabolic as well as anthelminic resistance are under positive selection.

BACKGROUND: The evolution of parasites is often directly affected by the host's environment. Studies on the evolution of the same parasites in different hosts are of great interest and are highly relevant to our understanding of divergence. METHODS: Here we performed whole-genome sequencing of Parascaris univalens from different Equus hosts (horses, zebras and donkeys). Phylogenetic and selection analyses were performed to study the divergence and adaptability of P. univalens. RESULTS: At the genetic level, multiple lines of evidence indicate that P. univalens is mainly separated into two clades (horse-derived and zebra & donkey-derived). This divergence began 300-1000 years ago, and we found that most of the key enzymes related to glycolysis were under strong positive selection in zebra & donkey-derived roundworms, whereas the lipid-related metabolic system was under positive selection in horse-derived roundworms, indicating that the adaptive evolution of metabolism has occurred over the past few centuries. In addition, we found that some drug-related genes showed a significantly higher degree of selection in diverse populations. CONCLUSIONS: This work reports the adaptive evolution and divergence trend of P. univalens in different hosts for the first time. Its results indicate that the divergence of P. univalens is a continuous, dynamic process. Furthermore, the continuous monitoring of the effects of differences in nutritional and drug histories on the rapid evolution of roundworms is conducive to further understanding host-parasite interactions.

Highly Pathogenic Avian Influenza A(H5N8) Clade 2.3.4.4b Viruses in Satellite-Tracked Wild Ducks, Ningxia, China, 2020.

During October 2020, we identified 13 highly pathogenic avian influenza A(H5N8) clade 2.3.4.4b viruses from wild ducks in Ningxia, China. These viruses were genetically related to H5N8 viruses circulating mainly in poultry in Europe during early 2020. We also determined movements of H5N8 virus‒infected wild ducks and evidence for spreading of viruses.

The first feline immunodeficiency virus from Siberian tigers (Panthera tigris altaica) in northeastern China.

Feline immunodeficiency virus (FIV) naturally infects more than 20 kinds of felines and poses a serious threat to their health, but there has been little research on FIV in tigers. In this study, 320 captive Siberian tigers (225 from Harbin, 55 from Hailin, and 40 from Shenyang) were tested for FIV by nested PCR, and three Siberian tigers from Hailin were FIV positive (5.45%). From these three animals, FIV gene fragments, gag-p26 (444 nt) from samples HD094 and HD1786 and pol-RT (576 nt) and pol-RNase (730 nt) from sample HD631, were sequenced and found to share more than 99% sequence identity with FIV subtype A from domestic cats. This is the first time FIV has been detected in Siberian tigers in China.

First isolation of Trichuris from Chinese serow, Capricornis milneedwardsii (Cetartiodactyla: Caprinae).

Whipworms are soil-transmitted helminths that can infect a variety of animals. A Chinese serow possibly infected by whipworms was found during a wildlife disease surveillance project in Baima Snow Mountain National Nature Reserve, Yunnan, China, in 2021. As convergent evolution is common in coinhabiting parasites, a mitochondrial gene sequence (cox1) and ribosomal gene sequence (ITS1) were used to identify species similar to Trichuris from Chinese serow. The phylogenetic trees and genetic distances of ten Trichuris samples from a Chinese serow together with other Trichuris spp. that have been previously reported were analysed based on the cox1 and ITS1 sequences. The combined results of the phylogenetic tree and genetic distances based on cox1 gene showed that the whipworms in Chinese serow are T. skrjabini. However, the whipworms in the present study were divided into two apparent clades in the phylogenic trees constructed by the cox1 sequences (Clades A and B) and the ITS1 sequences (Clades C and D). In addition, the Fst and Nm values were 0.82 and 0.23 between Clade A and Clade B for the cox1 gene, and 0.30 and 0.45 between Clade C and Clade D for the ITS1 sequences; both indicators showed low gene flow among the clades. Therefore, the genetic population structure of T. skrjabini was illustrated.

Highly Pathogenic Avian Influenza A(H5N8) Virus in Swans, China, 2020.

In October 2020, highly pathogenic avian influenza A(H5N8) viruses were detected in 2 dead swans in Inner Mongolia, China. Genetic analysis showed that the H5N8 isolates belong to clade 2.3.4.4b and that the isolates cluster with the H5N8 viruses isolated in Eurasia in the fall of 2020.

Ascarid infection in wild Amur tigers (Panthera tigris altaica) in China.

BACKGROUND: Wild Amur tigers are a sparsely populated species, and the conservation of this species is of great concern worldwide, but as an important health risk factor, parasite infection in them is not fully understanding. RESULTS: In this study, sixty-two faecal samples were collected to investigate the frequency and infection intensity of Toxocara cati and Toxascaris leonina in wild Amur tigers. The T. cati and T. leonina eggs were preliminary identified by microscopy, and confirmed by molecular techniques. Infection intensity was determined by the modified McMaster technique. Phylogenetic trees demonstrated that T. cati of wild Amur tiger had a closer relationship with which of other wild felines than that of domestic cats. T. leonina of Amur tiger and other felines clustered into one clade, showing a closer relationship than canines. The average frequency of T. cati was 77.42% (48/62), and the frequency in 2016 (100%) were higher than those in 2013 (P = 0.051, < 0.1; 66.6%) and 2014 (P = 0.079, < 0.1; 72.2%). The infection intensity of T. cati ranged from 316.6 n/g to 1084.1 n/g. For T. leonina, only three samples presented eggs when the saturated sodium chloride floating method was performed, indicating that the frequency is 4.83% (3/62). Unfortunately, the egg number in faecal smears is lower than the detective limitation, so the infection intensity of T. leonina is missed. CONCLUSIONS: This study demonstrated that ascarids are broadly prevalent, and T. cati is a dominant parasite species in the wild Amur tiger population.

New H6 influenza virus reassortment strains isolated from Anser fabalis in Anhui Province, China.

BACKGROUND: H6 subtype avian influenza viruses are globally distributed and, in recent years, have been isolated with increasing frequency from both domestic and wild bird species as well as infected humans. Many reports have examined the viruses in the context of poultry or several wild bird species, but there is less information regarding their presence in migratory birds. METHODS: Hemagglutination and hemagglutination inhibition tests were used to measure HA activity for different HA subtypes. Whole viral genomes were sequenced and analysed using DNAstar and MEGA 6 to understand their genetic evolution. Pathogenicity was evaluated using a mouse infection model. RESULTS: We isolated 13 strains of H6 virus from faecal samples of migratory waterfowl in Anhui Province of China in 2014. Phylogenetic analysis showed gene reassortment between Eurasian and North American lineages. Five of the identified H6 strains had the ability to infect mice without adaptation. CONCLUSION: Our findings suggest that regular surveillance of wild birds, especially migratory birds, is important for providing early warning and control of avian influenza outbreaks.

Experimental infection of dogs with H6N1 avian influenza A virus.

H6N1 avian influenza A viruses, which have spread across North America, Europe and Asia, have been shown to be infectious not only for birds but also for mammals. Because humans lack immunity to H6N1 avian influenza A viruses, the emergence of these viruses in humans would probably cause a pandemic. Replication of H6N1 avian influenza A viruses in dogs may facilitate their adaptation in humans because dogs are often in close contact with humans. However, the susceptibility of dogs to these viruses is unknown. To address this question, we infected beagles intranasally (i.n.) with an H6N1 avian influenza A virus that was isolated from a mallard. Inoculation of this virus into beagles resulted in the virus being detectable in the lung and seroconversion with no clinical signs except for a fever at 1 day post-inoculation (dpi). In addition, the virus was transiently shed from the nose and in the feces of the infected beagles. Our results suggest that dogs can be subclinically infected with H6N1 avian influenza A viruses, which, like H7N9, have low pathogenicity in birds and may serve as an intermediate host to transfer this virus to humans. Certain actions may be taken to prevent the potential transmission of these viruses, including the development of H6N1 avian influenza vaccines for prevention.

Genetic diversity and phylogenetic analysis of glycoprotein gp85 of avian leukosis virus subgroup J wild-bird isolates from Northeast China.

Avian leukosis virus subgroup J (ALV-J), first isolated in 1989, preferentially infects meat-type birds. Chinese layer flocks have experienced outbreaks of this virus since 2008. To analyze the status of ALV-J infection in wild birds in China, 585 wild birds collected from three provinces of Northeast China from 2010 to 2012 were tested, and six ALV-J strains were isolated for the first time. Furthermore, the gp85 genes of the six strains were amplified, cloned, and sequenced. The results indicated that two different ALV-J strains coexisted in Chinese wild birds from 2010 to 2012. These results not only expand the epidemiological data available for ALV-J and provide necessary information for the further understanding of the evolution of ALV-J, but they also highlight the potential role of wild-bird migration in the spread of ALV-J.

First isolation of reticuloendotheliosis virus from mallards in China.

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in many avian hosts worldwide. REV infection has never been reported in mallard ducks, however. To identify REV infection in mallards, we collected 40 mallard duck samples from Jilin Province of China. In this study, the REV strain, DBYR1102, was first isolated from a mallard in China and identified by PCR, indirect immunofluorescence assay and electron microscopy. The gp90 gene and complete LTR of DBYR1102 were amplified and sequenced. Phylogenetic analysis based on gp90 genes of REV indicated that the REV strain DBYR1102 is closely related to strain HLJR0901 from northeastern China, the prairie chicken isolate APC-566, and REV subtype III, represented by chick syncytial virus. This new strain is distantly related to two other subtypes of REV, 170A and SNV. Phylogenetic analysis based on the LTR yielded information similar to that obtained with the gp90 genes. The results of this study not only expand our epidemiological understanding of REV in the wild birds of China but also demonstrate the potential role of wild waterfowl in REV transmission.

Complete nucleotide sequence of avian paramyxovirus type 6 strain JL isolated from mallard ducks in China.

A new strain of avian paramyxovirus type 6 (APMV-6), JL, has been isolated from mallard ducks in China, and its complete genome has been sequenced and analyzed. This work is the first announced complete genome sequence of APMV-6 from mallards.

H10Nx avian influenza viruses detected in wild birds in China pose potential threat to mammals.

H10 subtype avian influenza viruses (AIVs) have been isolated from wild and domestic avian species worldwide and have occasionally crossed the species barrier to mammalian hosts. Fatal human cases of H10N8 infections and the recent detection of human H10N3 infections have drawn widespread public attention. In this study, 25 H10Nx viruses were isolated from wild waterfowl in China during a long-term surveillance of AIVs. We conducted phylogenetic and phylogeographic studies of the hemagglutinin (HA) genes of global H10 viruses to determine the spatiotemporal patterns of spread and the roles of different hosts in viral transmission. We found the pattern of AIV transmission from wild birds to poultry to humans, and Anatidae have acted as the seeding population in the spread of the virus. Phylogenetic incongruence indicated complex reassortment events and our isolates were divided into eight genotypes (G1-8). We also found that the HA genes of the G8 viruses belonged to the North American lineage, indicating that intercontinental gene flow has occurred. Their receptor-binding specificity showed that the G1/4/5/6/7/8 viruses bind to both human-type α2,6-linked sialic acid receptors and avian-type α2,3-linked sialic acid receptors. Mouse studies indicated that the H10Nx isolates replicated efficiently in the respiratory system without preadaptation, but showed low pathogenicity in mice. The H10Nx isolates showed no (G2/4/7) or low pathogenicity (G1/3/5/6/8) in chickens, and the G6 and G8 viruses could be transmitted to chickens through direct contact. The asymptomatic shedding of these wild-bird-origin H10Nx isolates in chickens and their good adaptation in mice should increase the ease of their transmission to humans, and they therefore pose a threat to public health. Our findings demonstrate a further understanding of wild bird-origin H10 viruses and provide information for the continuous surveillance of H10 subtype viruses.

Per- and polyfluoroalkyl substances (PFASs) in the blood of police and Beagle dogs from Harbin, China: Concentrations and associations with hematological parameters.

Per-and polyfluoroalkyl substances (PFASs) have been omnipresent in the environment and marine organisms. However, little is known about these compounds and their associations with hematological parameters in dogs. In this research, we investigated the concentrations and distributions of PFASs in the blood of dogs and explored the associations between PFASs concentrations in blood and hematological parameters. Perfluorooctanoate (PFOA) was found to be the dominant PFAS in the blood (54.23%), followed by perfluorobutyric acid (PFBA) (16.05%) and perfluorooctanesulfonate (PFOS) (12.05%). On average, PFASs concentration in dogs was 3.553 ng/mL (SD: 2.146). Moreover, age is a key factor influencing the levels of PFBA, PFOA, and PFBS in males, as well as seven PFASs (6:2 Cl-PFESA, PFBA, PFOA, PFOS, PFHxS, PFDA, and PFNA) in female dogs' blood. The results revealed that PFHxS in dietary food accounted for most of the total daily PFASs consumption. We also discovered that greater PFASs exposure (including PFOA and PFOS) could significantly increase amylase (AMY) and decrease cholesterol (CHOL) levels. Furthermore, there are linear relationships between PFDA, PFNA and many biochemical parameters (AMY, CHOL, albumin/globulin (A/G), blood urea nitrogen (BUN), alkaline phosphatase (ALP), creatinine (CREA)). Thus, PFAS accumulation has a certain influence on dogs' health, and we must pay attention to the potential threat posed by these elements to dogs.

Molecular Detection of Parvovirus in Captive Siberian Tigers and Lions in Northeastern China From 2019 to 2021.

The fact that wild felines are carriers of pernicious infectious viruses should be a major concern due to the potential cross-species transmission between the felines and human or domestic animals. However, studies on the virus in the captive wild felines, especially in tigers, are thin on the ground. In this study, we screened four infectious viruses, namely, feline parvovirus (FPV), feline coronavirus (FCoV), canine distemper virus (CDV), and influenza A virus (IAV), in the blood samples of 285 captive Siberian tigers (Panthera tigris altaica) and in the spleen samples of two deceased lions (Panthera leo), which were collected from 2019 to 2021 in three Siberian Tiger Parks from the northeast of China. Nucleic acids isolated from the blood samples collected from tigers and the spleen samples collected from two deceased lions were positive for FPV by PCR, and the positive rate was 4.6% (13/285) in tigers. Furthermore, the VP2 gene of FPV was amplified by nested PCR, and the sequences of the VP2 gene from these six FPV positive strains shared 98.3-99.9% homology with the reference. The key amino acid sites of VP2 protein were consistent with that of FPV reference strains. Phylogenetic analysis based on the VP2 gene showed that in this study, FPV-positive strains were grouped within the FPV clade and closely related to the Asian strains clade. The results of this study showed that FPV circulated in the captive Siberian tigers and lions in northeastern China and provided valuable information for the study of FPV epidemiology in wild felines. Therefore, we suggest that regular antibody monitoring and booster immunization for tigers should be performed.

Prevalence of four viruses in captive Siberian tigers from Northeastern China.

Our study investigated the prevalence of feline herpesvirus-1 (FHV-1), feline calicivirus (FCV), feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV) in captive Siberian tigers in Northeastern China. A total of 324 blood samples and 33 nasopharyngeal swab samples of Siberian tigers collected from 2019 to 2021 in three cities were investigated by nested PCR. The results showed that 28.1% (91/324) tigers were infected with at least one virus; the positive rates of FHV-1, FCV, and FIV were 17.3%, 13.6%, and 0.9%, respectively; and the coinfection prevalence was 13.2%. No FeLV-positive sample was detected. And we found that the blood is the best for FCV, FIV, and FeLV detection, but nasopharyngeal swabs for FHV-1. By comparing the gD genes, TK gene, and gI gene of FHV-1, the homology of the three FHV-1 positive strains in this study was found to be 91.5%-99.9% shared with tigers and domestic cats. Based on a comparison of the nucleic acid sequences of 13 FCV strains, we found that the homology of strain HB-1926 with the other strains in this study was only about 77.7%, but shared 99.3% and 98.6% homology with Urnaba strain in American cat and TG1 strain in Chinese tiger, respectively. However, the other 12 FCV strains shared 87.1%-87.5% homology compared with the Chinese domestic cats. Phylogenetic tree analysis showed that the HB-1926 strain was not in the same clade as other strains. The fragments gag-p26, pol-RT, and pol-RNAse of Siberian tiger FIV shared more than 99% homology than domestic cats FIV subtype A. This study demonstrated that captive Siberian tigers in Northeastern China were exposed to FHV-1, FCV, and FIV, and it is necessary to develop more effective vaccines and improve daily management measures.

Cross-species transmission of feline herpesvirus 1 (FHV-1) to chinchillas.

BACKGROUND: Herpesviruses are a class of double-stranded DNA viruses found in both vertebrates and invertebrates. They are usually highly host-specific and do not easily spread across species. Chinchillas have gradually entered the Chinese pet market in recent years, but references to viral infections in chinchillas are extremely scarce, and only two reports about the herpesvirus in chinchillas are available at present. OBJECTIVES: The aim of this study was to present the first report of FHV-1 infection in chinchillas. METHODS: A total of 130 nasopharyngeal swab samples of chinchillas and three nasopharyngeal swabs of domestic cats collected from a chinchillas farm were investigated by nested PCR for FHV-1. RESULTS: Four chinchillas were infected with FHV-1, the positive rate was 3.08% (4/130), and two domestic cats were FHV-1 positive (2/3). The 253 bp fragments of FHV-1 gD gene from four chinchillas and two domestic cats were 100% identical, respectively, and the homology between chinchillas and domestic cat was 99.21%, but they all shared nearly 98.81% homology with the reference strain sequences. Phylogenetic tree analysis showed that these four chinchillas strains were clustered together with FHV-1. CONCLUSIONS: This is the first time that FHV-1 was detected in chinchillas and suggested chinchillas are susceptible to FHV-1 and may play a role as a temporary reservoir for FHV-1.

The Variation of Duck RIG-I-Mediated Innate Immune Response Induced by Different Virulence Avian Influenza Viruses.

In recent years, the emerging highly pathogenic avian influenza (HPAI) A(H5N8) virus has been reported with features of widely spread, an expanding host range, and cross-species transmission, attracting wide attention. The domestic duck plays a major role in the epidemiological cycle of the HPAI H5N8 virus, but little is known concerning innate immune responses during influenza infection in duck species. In this study, we used two wild-bird-origin viruses, H5N8 and H4N6, to conduct duck infection experiments, and detect the load of the two viruses, and retinoic acid-inducible gene I (RIG-I) and interferon ß (IFN-ß) in the host's natural immune response. Through comparison, it is found that the expression levels of RIG-I and IFN-ß are both fluctuating. The innate immunity starts rapidly within 6 h after infection and is inhibited by the virus to varying degrees. The expression of RIG-I and IFN-ß decreased on 1-2 days post-infection (dpi). The HPAI H5N8 virus has a stronger inhibitory effect on RIG-I than the low pathogenic avian influenza (LPAI) H4N6 virus and is the strongest in the lungs. After infection with HPAI H5N8 virus, 2 dpi, viral RNA replicates in large amounts in the lungs. It has been proven that RIG-I and IFN-ß play an important role in the innate immune response of ducks to HPAI H5N8 virus infection, especially in the lungs. The main battlefield of RIG-I and IFN-ß after infection with the LPAI H4N6 virus is in the rectum. Both viruses have been effectively controlled after 7 dpi. These results will help to understand the transmission mechanisms of avian influenza virus in wild ducks and help effectively prevent and control avian influenza.