Animal Infection Experiments Research Articles

Pathogenicity of Multidrug-Resistant Salmonella typhimurium Isolated from Ducks.

Salmonella typhimurium (S. typhimurium) is one of the most common Salmonella serotypes in epidemiological surveys of poultry farms in recent years. It causes growth retardation, mortality, and significant economic losses. The extensive use of antibiotics has led to the emergence of multi-drug resistance (MDR) in Salmonella, which has become a significant global problem and long-term challenge. In this study, we investigated the prevalence and features of S. typhimurium strains in duck embryos and cloacal swabs from large-scale duck farms in Shandong, China, including drug resistance and virulence genes and the pathogenicity of an S. typhimurium strain by animal experiment. The results demonstrated that a total of 8 S. typhimurium strains were isolated from 13,621 samples. The drug resistance results showed that three of the eight S. typhimurium strains were MDR with the dominant resistance profile of CTX-DX-CTR-TE-AMX-AMP-CAZ. In particular, the virulence genes invA, hilA, pefA, rck, and sefA showed high positive rates. Based on the analysis of the biological characteristics of bacterial biofilm formation and mobility, a strain of S. typhimurium with the strongest biofilm formation ability, designated 22SD07, was selected for animal infection experiments with broiler ducklings. The results of animal experiments demonstrated that infection with 22SD07 reduced body weight and bursa index but increased heart and liver indexes compared to the control group. Histological examination revealed desquamation of the intestinal villous epithelium, the presence of large aggregates of lymphocytes, and a decrease in goblet cells following infection. Furthermore, the expression of IL-10 was significantly increased in the liver at 3 dpi, while TNF-α was significantly increased in the spleen at 7 dpi. The above results indicate that S. typhimurium may pose a potential threat to human health through the food chain. This helps us to understand the frequency and characteristics of S. typhimurium in duck farms and emphasizes the urgent need to strengthen and implement effective continuous monitoring to control its infection and transmission.

A c-di-GMP binding effector STM0435 modulates flagellar motility and pathogenicity in Salmonella

ABSTRACT Flagella play a crucial role in the invasion process of Salmonella and function as a significant antigen that triggers host pyroptosis. Regulation of flagellar biogenesis is essential for both pathogenicity and immune escape of Salmonella. We identified the conserved and unknown function protein STM0435 as a new flagellar regulator. The ∆stm0435 strain exhibited higher pathogenicity in both cellular and animal infection experiments than the wild-type Salmonella. Proteomic and transcriptomic analyses demonstrated dramatic increases in almost all flagellar genes in the ∆stm0435 strain compared to wild-type Salmonella. In a surface plasmon resonance assay, purified STM0435 protein-bound c-di-GMP had an affinity of ~8.383 µM. The crystal structures of apo-STM0435 and STM0435&c-di-GMP complex were determined. Structural analysis revealed that R33, R137, and D138 of STM0435 were essential for c-di-GMP binding. A Salmonella with STM1987 (GGDEF protein) or STM4264 (EAL protein) overexpression exhibits completely different motility behaviours, indicating that the binding of c-di-GMP to STM0435 promotes its inhibitory effect on Salmonella flagellar biogenesis.

Survey of the Prosthogonimus spp. metacercariae infection in the second intermediate host dragonfly in Heilongjiang Province, China.

Prosthogonimiasis poses a threat to the reproductive system of poultry and wild birds, which are the definitive hosts of the parasite causing this disease. However, the parasite infection of the second intermediate host (dragonfly), the primary vector of this pathogen, is rarely reported. In this study, the prevalence of Prosthogonimus infection in dragonflies was investigated from June 2019 to October 2022 in Heilongjiang Province, northeast China. The species of metacercariae isolated from dragonfly were identified by morphological characteristics, molecular biology techniques, and animal infection experiments. The results showed that 11 species of dragonflies and one damselfly were identified and among six of the dragonflies infected by Prosthogonimus metacercariae, Sympetrum depressiusculum (28.53%) had the highest infection rate among all positive dragonflies, followed by Sympetrum vulgatum (27.86%) and Sympetrum frequens (20.99%), which are preferred hosts, and the total prevalence was 20.39% (2061/10,110) in Heilongjiang Province. Three species of Prosthogoniumus metacercariae were isolated, including Prosthogonimus cuneatus, Prosthogonimus pullucidus, and Prosthogonimus sp., among which P. cuneatus was the dominant species in dragonflies in Heilongjiang Province. This is the first report on the prevalence of Prosthogonimus in dragonflies in China, which provides baseline data for the control of prosthogonimiasis in Heilongjiang Province and a reference for the prevention of prosthogonimiasis in other areas of China.

Animal host range of mpox virus.

Mpox is caused by the mpox virus, which belongs to the Orthopoxvirus genus and Poxviridae family. Animal hosts, such as African rodents, mice, prairie dogs, and non-human primates, play important roles in the development and transmission of outbreaks. Laboratory animal infection experiments have demonstrated that some animals are susceptible to mpox virus. This review summarizes the current progress on the animal hosts for mpox virus. The surveillance of mpox virus in animal hosts will provide important insights into virus tracing, analysis of mutation evolutionary patterns, transmission mechanisms, and development of control measures.

A homolog of low molecular weight protein tyrosine phosphatase isolated from Brucella melitensis displays an acidic dual specific phosphatase activity, nonessential for bacterial resistance to bactericidal factors and virulence

Brucellosis is a bacterial infectious zoonosis which is spread worldwide, caused by Brucella, with infertility and abortion in domestic animals. Protein-tyrosine phosphatase (PTPs) have been discovered in many kinds of bacterial species, which play crucial roles in many aspects, such as bacterial physiology and virulence. However, no PTPs have been identified in Brucella to date. Here, we identified a novel gene BM28_RS15985 in Brucella melitensis that encodes a homolog of a low weight molecular PTP. Enzyme activity analysis showed that this PTP is a dual specific phosphatase, removing phosphate group from phosphotyrosine and phosphoserine/phosphothreonine peptides, which was designated as Dsp1. The optimal pH of the Dsp1 enzyme activity were 5.5, suggesting that the Dsp1 is an acidic phosphatase, and the optimal reaction temperature of the Dsp1 was 35.0 °C. Besides, the Michaelis constant and maximum reaction velocity of the Dsp1 were 40.17 mM and 24.33 nM/min/mg, respectively. In further study, we investigated the role of Dsp1 in B. melitensis phenotype and virulence. Growth curve and resistance test exhibited that the dsp1 had no role in Brucella growth and resisting bactericidal factors. Cell and animal infection experiment showed that the dsp1 deletion did not affect the intracellular survival and virulence of B. melitensis. In summary, we identified a novel acidic dual specific phosphatase in B. melitensis and evaluated its characteristics of the enzyme activity, this study will expand the understanding of Brucella phosphatase.

Genomic Sequence and Pathogenicity of the Chicken Anemia Virus Isolated From Chicken in Yunnan Province, China.

Chicken anemia virus (CAV), which has been reported in many countries, causes severe anemia and immunosuppression in chickens. In this study, a CAV strain YN04 belonging to genotype A was first identified from infected chickens in Yunnan province, China. Moreover, the animal infection experiments further confirmed that the strain YN04 is a highly pathogenic strain, which can cause 86.67% mortality in chickens in the infection group. The mean death time of infected chickens was 13.1 days post infection (dpi). CAV infection induced severe anemia with significant decrease in packed cell volume (PCV), and serious atrophy and lesion of thymus and bursa with high viral load at 14 dpi. Besides, CAV infection caused a sharp decrease in chicken body weight and immune organ indices including the ratio of thymus or bursa to body weight at 21 dpi, which displayed the potential immunosuppression state at this stage. These findings enrich the epidemiological data on CAV and may provide information for preventing its further spread in Yunnan province, China.

Pathogenesis and genetic characteristics of a novel reassortant low pathogenic avian influenza A(H7N6) virus isolated in Cambodia in 2019.

The first human case of zoonotic A(H7N4) avian influenza virus (AIV) infection was reported in early 2018 in China. Two months after this case, novel A(H7N4) viruses phylogenetically related to the Jiangsu isolate emerged in ducks from live bird markets in Cambodia. During active surveillance in Cambodia, a novel A(H7N6) reassortant of the zoonotic low pathogenic AIV (LPAIV) A(H7N4) was detected in domestic ducks at a slaughterhouse. Complete genome sequencing and phylogenetic analysis showed that the novel A(H7N6) AIV is a reassortant, in which four gene segments originated from Cambodia A(H7N4) viruses and four gene segments originated from LPAIVs in Eurasia. Animal infection experiments revealed that chickens transmitted the A(H7N6) virus via low-level direct contacts, but ducks did not. Although avian-origin A(H7Nx) LPAIVs do not contain the critical mammalian-adaptive substitution (E627K) in PB2, the lethality and morbidity of the A(H7N6) virus in BALB/c mice were similar to those of A(H7N9) viruses, suggesting potential for interspecies transmission. Our study reports the emergence of a new reassortant of zoonotic A(H7N4) AIVs with novel viral characteristics and emphasizes the need for ongoing surveillance of avian-origin A(H7Nx) viruses.

Pathogenicity and transmissibility of a novel respirovirus isolated from a Malayan pangolin.

The identification of SARS-CoV-2-like viruses in Malayan pangolins (Manis javanica) has focused attention on these endangered animals and the viruses they carry. We successfully isolated a novel respirovirus from the lungs of a dead Malayan pangolin. Similar to murine respirovirus, the full-length genome of this novel virus was 15 384 nucleotides comprising six genes in the order 3'-(leader)-NP-P-M-F-HN-l-(trailer)-5'. Phylogenetic analysis revealed that this virus belongs to the genus Respirovirus and is most closely related to murine respirovirus. Notably, animal infection experiments indicated that the pangolin virus is highly pathogenic and transmissible in mice, with inoculated mice having variable clinical symptoms and a fatality rate of 70.37 %. The virus was found to replicate in most tissues with the exception of muscle and heart. Contact transmission of the virus was 100 % efficient, although the mice in the contact group displayed milder symptoms, with the virus mainly being detected in the trachea and lungs. The isolation of a novel respirovirus from the Malayan pangolin provides new insight into the evolution and distribution of this important group of viruses and again demonstrates the potential infectious disease threats faced by endangered pangolins.

Host restriction of emerging high-pathogenic bunyaviruses via MOV10 by targeting viral nucleoprotein and blocking ribonucleoprotein assembly.

Bunyavirus ribonucleoprotein (RNP) that is assembled by polymerized nucleoproteins (N) coating a viral RNA and associating with a viral polymerase can be both the RNA synthesis machinery and the structural core of virions. Bunyaviral N and RNP thus could be assailable targets for host antiviral defense; however, it remains unclear which and how host factors target N/RNP to restrict bunyaviral infection. By mass spectrometry and protein-interaction analyses, we here show that host protein MOV10 targets the N proteins encoded by a group of emerging high-pathogenic representatives of bunyaviruses including severe fever with thrombocytopenia syndrome virus (SFTSV), one of the most dangerous pathogens listed by World Health Organization, in RNA-independent manner. MOV10 that was further shown to be induced specifically by SFTSV and related bunyaviruses in turn inhibits the bunyaviral replication in infected cells in series of loss/gain-of-function assays. Moreover, animal infection experiments with MOV10 knockdown corroborated the role of MOV10 in restricting SFTSV infection and pathogenicity in vivo. Minigenome assays and additional functional and mechanistic investigations demonstrate that the anti-bunyavirus activity of MOV10 is likely achieved by direct impact on viral RNP machinery but independent of its helicase activity and the cellular interferon pathway. Indeed, by its N-terminus, MOV10 binds to a protruding N-arm domain of N consisting of only 34 amino acids but proving important for N function and blocks N polymerization, N-RNA binding, and N-polymerase interaction, disabling RNP assembly. This study not only advances the understanding of bunyaviral replication and host restriction mechanisms but also presents novel paradigms for both direct antiviral action of MOV10 and host targeting of viral RNP machinery.

Evaluation of the immune response in conventionally weaned pigs infected with porcine deltacoronavirus.

Although porcine deltacoronavirus (PDCoV) is a significant pandemic threat in the swine population and has caused significant economic losses, information regarding the immune response in conventionally weaned pigs infected with PDCoV is scarce. Hence, the immune response in conventionally weaned pigs infected with PDCoV was assessed after challenge and rechallenge. After the first challenge, obvious diarrhea and viral shedding developed successively in all pigs in the four inoculation dose groups from 3 to 14 days postinfection (dpi), and all pigs recovered (no clinical symptoms or viral shedding) by 21 dpi. All pigs in the four groups exhibited significantly increased PDCoV-specific IgG, IgA and virus-neutralizing (VN) antibody (Ab) titers and IFN-γ levels in the serum after the first challenge. All pigs were completely protected against rechallenge at 21 dpi. The serum levels of PDCoV-specific IgG, IgA, and VN Abs increased further after rechallenge. Notably, the IFN-γ level declined continuously after 7 dpi. In addition, the levels of PDCoV-specific IgG, IgA and VN Abs in saliva increased significantly after rechallenge and correlated well with the serum Ab titers. Furthermore, the appearance of clinical symptoms of PDCoV infection in conventionally weaned pigs was delayed with reduced inoculation doses. In summary, the data presented here offer important reference information for future PDCoV animal infection and vaccine-induced immunoprotection experiments.

Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage.

Using bacteriophage-derived endolysins as an alternative strategy for fighting drug-resistant bacteria has recently been garnering renewed interest. However, their application is still hindered by their narrow spectra of activity. In our previous work, we demonstrated that the endolysin LysIME-EF1 possesses efficient bactericidal activity against multiple strains of Enterococcus faecalis (E. faecalis). Herein, we observed an 8 kDa fragment and hypothesized that it contributes to LysIME-EF1 lytic activity. To examine our hypothesis, we determined the structure of LysIME-EF1 at 1.75 Å resolution. LysIME-EF1 exhibits a unique architecture in which one full-length LysIME-EF1 forms a tetramer with three additional C-terminal cell-wall binding domains (CBDs) that correspond to the abovementioned 8 kDa fragment. Furthermore, we identified an internal ribosomal binding site (RBS) and alternative start codon within LysIME-EF1 gene, which are demonstrated to be responsible for the translation of the truncated CBD. To elucidate the molecular mechanism for the lytic activity of LysIME-EF1, we combined mutagenesis, lytic activity assays and in vivo animal infection experiments. The results confirmed that the additional LysIME-EF1 CBDs are important for LysIME-EF1 architecture and its lytic activity. To our knowledge, this is the first determined structure of multimeric endolysin encoded by a single gene in E. faecalis phages. As such, it may provide valuable insights into designing potent endolysins against the opportunistic pathogen E. faecalis.

Galleria mellonella as an alternative in vivo model to study bacterial biofilms on stainless steel and titanium implants

The purpose of this study was to establish an infection model of Galleria mellonella larvae as an alternative in vivo model for biofilm-associated infections on stainless steel and titanium implants. First, the model was established with sterile implants to evaluate biocompatibility. Titanium or stainless steel implants were implanted without adverse effects over the entire observation period of 5 days compared to controls and even up to the pupae and moth stage. Then, stainless steel and titanium implants contaminated with Staphylococcus aureus were implanted into larvae to mimic biofilm-associated infection. For both materials, pre-incubation of the implant with S. aureus led to significantly reduced survival of the larvae compared to sterile implants. Larvae could not be rescued by gentamicin, whereas gentamicin significantly improved the survival of the larvae in case of planktonic infection with S. aureus without an implant, confirming the typical character­istics of reduced antibiotic susceptibility of biofilm infections. Biofilm formation and various stages of biofilm maturation were confirmed by surface electron microscopy and by measuring bacterial gene expression of biofilm-related genes on contaminated implants, which confirmed biofilm formation and upregulation of autolysin (atl ) and sarA genes. In con­clusion, G. mellonella can be used as an alternative in vivo model to study biofilm-associated infections on stainless steel and titanium implants, which may help to reduce animal infection experiments with vertebrates in the future.

Galleria mellonella as an alternative in vivo model to study bacterial biofilms on stainless steel and titanium implants_suppl2

The purpose of this study was to establish an infection model of Galleria mellonella larvae as an alternative in vivo model for biofilm-associated infections on stainless steel and titanium implants. First, the model was established with sterile implants to evaluate biocompatibility. Titanium or stainless steel implants were implanted without adverse effects over the entire observation period of 5 days compared to controls and even up to the pupae and moth stage. Then, stainless steel and titanium implants contaminated with Staphylococcus aureus were implanted into larvae to mimic biofilm-associated infection. For both materials, pre-incubation of the implant with S. aureus led to significantly reduced survival of the larvae compared to sterile implants. Larvae could not be rescued by gentamicin, whereas gentamicin significantly improved the survival of the larvae in case of planktonic infection with S. aureus without an implant, confirming the typical character­istics of reduced antibiotic susceptibility of biofilm infections. Biofilm formation and various stages of biofilm maturation were confirmed by surface electron microscopy and by measuring bacterial gene expression of biofilm-related genes on contaminated implants, which confirmed biofilm formation and upregulation of autolysin (atl ) and sarA genes. In con­clusion, G. mellonella can be used as an alternative in vivo model to study biofilm-associated infections on stainless steel and titanium implants, which may help to reduce animal infection experiments with vertebrates in the future.

Galleria mellonella as an alternative in vivo model to study bacterial biofilms on stainless steel and titanium implants_suppl1

The purpose of this study was to establish an infection model of Galleria mellonella larvae as an alternative in vivo model for biofilm-associated infections on stainless steel and titanium implants. First, the model was established with sterile implants to evaluate biocompatibility. Titanium or stainless steel implants were implanted without adverse effects over the entire observation period of 5 days compared to controls and even up to the pupae and moth stage. Then, stainless steel and titanium implants contaminated with Staphylococcus aureus were implanted into larvae to mimic biofilm-associated infection. For both materials, pre-incubation of the implant with S. aureus led to significantly reduced survival of the larvae compared to sterile implants. Larvae could not be rescued by gentamicin, whereas gentamicin significantly improved the survival of the larvae in case of planktonic infection with S. aureus without an implant, confirming the typical character­istics of reduced antibiotic susceptibility of biofilm infections. Biofilm formation and various stages of biofilm maturation were confirmed by surface electron microscopy and by measuring bacterial gene expression of biofilm-related genes on contaminated implants, which confirmed biofilm formation and upregulation of autolysin (atl ) and sarA genes. In con­clusion, G. mellonella can be used as an alternative in vivo model to study biofilm-associated infections on stainless steel and titanium implants, which may help to reduce animal infection experiments with vertebrates in the future.

Time-resolved dual RNA-seq of tissue uncovers Pseudomonas plecoglossicida key virulence genes in host-pathogen interaction with Epinephelus coioides.

Bacterial pathogen-host interactions are highly dynamic, regulated processes that have been primarily investigated using in vitro assays. The dynamics of bacterial pathogen-host interplay in vivo are poorly understood. Using time-resolved dual RNA-seq in a Pseudomonas plecoglossicida-Epinephelus coioides infection model, we observed that bacterial genes encoding classical virulence factors and host genes involved in immune regulation were dynamically expressed during infection. Using network inferencing, we were able to predict interspecies regulatory networks linking bacterial virulence genes to host immune genes. Together with gene co-expression network analysis of the pathogen, secY was predicted to be a key virulence gene for P. plecoglossicida pathogenicity in the host, fliN was predicted to be a less important virulence gene. The results of bioinformatics prediction were confirmed by animal infection experiments. Our work provides the first paradigm to study dynamic alterations of bacterial pathogen and host interactions based on the elucidation of time-resolved interactive transcriptomes in vivo, and may be developed into a novel and universal method for revealing the true complexity of the bacterial infection process.

Clinical and Serological Evaluation of LINDA Virus Infections in Post-Weaning Piglets.

The novel pestivirus species known as lateral-shaking inducing neuro-degenerative agent (LINDA) virus emerged in 2015 in a piglet-producing farm in Austria. Affected piglets showed strong congenital tremor as a result of severe lesions in the central nervous system. Here, we report the results of a controlled animal infection experiment. Post-weaning piglets were infected with LINDA to determine the susceptibility of pigs, the clinical consequences of infection and the humoral immune response against LINDA. No clinically overt disease signs were observed in the piglets. Viremia was hardly detectable, but LINDA was present in the spleen and several lymphatic organs until the end of the experiment on day 28 post-infection. Oronasal virus shedding together with the infection of one sentinel animal provided additional evidence for the successful replication and spread of LINDA in the piglets. Starting on day 14 post-infection, all infected animals showed a strong humoral immune response with high titers of neutralizing antibodies against LINDA. No cross-neutralizing activity of these sera with other pestiviral species was observed. According to these data, following postnatal infection, LINDA is a rather benign virus that can be controlled by the pig’s immune system. However, further studies are needed to investigate the effects of LINDA on the fetus after intrauterine infection.

MiR-125b Suppression Inhibits Apoptosis and Negatively Regulates Sema4D in Avian Leukosis Virus-Transformed Cells.

Subgroup J avian leukosis virus (ALV-J), an oncogenic retrovirus, causes hemangiomas and myeloid tumors in chickens. We previously showed that miR-125b is down-regulated in ALV-J-induced tumors. This study aimed to investigate the possible role of miR-125b in ALV-J-mediated infection and tumorigenesis. Knockdown of miR-125b expression in HP45 cells reduced, whereas over-expression induced late-stage apoptosis. Bioinformatics analysis and luciferase activity assays indicate that miR-125b targets Semaphorin 4D/CD100 (Sema4D) by binding the 3′-untranslated region of messenger RNA (mRNA). Up-regulation of miR-125b in the DF1 cell line suppressed Sema4D expression, whereas miR-125 down-regulation increased Sema4D expression levels. To uncover the function of Sema4D during ALV-J infection, animal infection experiments and in vitro assays were performed and show that Sema4D mRNA levels were up-regulated in ALV-J-infected tissues and cells. Finally, functional experiments show that miR-125 down-regulation and Sema4D over-expression inhibited apoptosis in HP45 cells. These results suggest that miR-125b and its target Sema4D might play an important role in the aggressive growth of HP45 cells induced by avian leukosis viruses (ALVs). These findings improve our understanding of the underlying mechanism of ALV-J infection and tumorigenesis.

Identification of six novel capsular polysaccharide loci (NCL) from Streptococcussuis multidrug resistant non-typeable strains and the pathogenic characteristic of strains carrying new NCLs.

Streptococcussuis is a major swine pathogen and an important zoonotic agent worldwide. At least nine serotypes can infect human so far. Although 29 serotypes (1-19, 21, 23-25, 27-31 and 1/2) strains are considered as authentic S.suis, a novel variant serotype Chz and strains carrying 20 novel capsular polysaccharide loci (NCL) have been identified recently. However, information about pathogenic and antimicrobial resistance characteristics of strains carrying NCLs is still unavailable. In this study, we identified six new NCLs (designated as NCL21-26) from 35 non-typeable S.suis strains by agglutination tests and whole genome sequencing analysis. Further analysis of the genetic context of NCL25 and NCL26 showed a mosaic structure of the capsular polysaccharide loci. NCL25 exhibited considerable similarity to that of serotypes 10 and 11, and NCL26 shared similarity to that of serotype 9 and NCL4. Antimicrobial susceptibility testing demonstrated that strains carrying NCL21-26 were all resistant to clindamycin, lincomycin, erythromycin, tilmicosin and tetracycline. Animal infection experiments showed that the virulence of NCL26 strain NJ1112 isolated from a disease pig was similar to that of S.suis serotype 2 virulent strain SC070731 in both zebrafish and mouse infection models, highlighting the necessity for surveillance of strains belonging to NCL26. We also developed a multiplex PCR assay to detect NCL21-26 strains. Our findings expand the views of genetic diversity of S.suis capsular polysaccharide loci and S.suis pathogenic characteristic.

Genotyping and pathogenic characterization of canine distemper virus based on mutations in the hemagglutinin gene in Chinese domestic dogs.

Canine distemper virus (CDV) infects wild and domestic Canidae worldwide. The hemag- glutinin (H) gene has the highest genetic variation in the genome of this virus. Thus, the H gene is commonly used for lineage identification and genetic analyses. In order to study the genetic characteristics and pathogenicity of CDV strains prevalent in China, 132 samples were collected from domestic dogs with suspected CDV infection, 58 samples were confirmed to be positive, and the H gene was successfully amplified from 15 samples. The epidemic strain was identified as type Asia-1 and the novel mutations, A51T, V58I, R179K and D262N, were detected in this strain. Isolated strains, BJ16B53, BJ16B14, and BJ17B8, were used for an animal infection experiment in raccoon dogs. BJ16B53 and BJ16B14 were found to cause clinical symptoms, death, and exten- sive lesions in various organs. These results are expected to facilitate the development of effective strategies to monitor and control CDV infection in China.

Two genetically diverse H7N7 avian influenza viruses isolated from migratory birds in central China

After the emergence of H7N9 avian influenza viruses (AIV) in early 2013 in China, active surveillance of AIVs in migratory birds was undertaken, and two H7N7 strains were subsequently recovered from the fresh droppings of migratory birds; the strains were from different hosts and sampling sites. Phylogenetic and sequence similarity network analyses indicated that several genes of the two H7N7 viruses were closely related to those in AIVs circulating in domestic poultry, although different gene segments were implicated in the two isolates. This strongly suggested that genes from viruses infecting migratory birds have been introduced into poultry-infecting strains. A Bayesian phylogenetic reconstruction of all eight segments implied that multiple reassortments have occurred in the evolution of these viruses, particularly during late 2011 and early 2014. Antigenic analysis using a hemagglutination inhibition test showed that the two H7N7 viruses were moderately cross-reactive with H7N9-specific anti-serum. The ability of the two H7N7 viruses to remain infectious under various pH and temperature conditions was evaluated, and the viruses persisted the longest at near-neutral pH and in cold temperatures. Animal infection experiments showed that the viruses were avirulent to mice and could not be recovered from any organs. Our results indicate that low pathogenic, divergent H7N7 viruses circulate within the East Asian-Australasian flyway. Virus dispersal between migratory birds and domestic poultry may increase the risk of the emergence of novel unprecedented strains.