An overview of SARS-CoV2 natural infections in companion animals: A systematic review of the current evidence.

This systematic review provides a comprehensive overview of natural SARS-CoV-2 infections in companion animals. The findings show that these infections are relatively rare. Among the examined dogs, only 1.32% tested positive for SARS-CoV-2, while for cats, the rate was 1.55%. Infections in rabbits and ferrets were even less common, at less than 1%. These results support previous research indicating the infrequency of natural infections in companion animals. The review also includes updated studies that involved various pets, such as cats, dogs, ferrets, and rabbits. The majority of the studies analyzed were primarily concerned with screening pets that visited veterinary clinics, regardless of whether they showed any specific signs of SARS-CoV-2 infection. Only a limited number of studies investigated infections in animals suspected of being in contact with owners or other animals that had COVID-19 or were exhibiting symptoms. The most common variant identified among the SARS-CoV-2 variants in the reviewed studies was B.1.1.7 (alpha), followed by B.1.617.2 (delta), B.1.526 (Iota), and others. The emergence of these variants raises concerns about their potential for increased transmissibility and virulence, highlighting the importance of ongoing monitoring of SARS-CoV-2 infections in both humans and animals. Furthermore, most of the reviewed studies indicated that infected pets either showed no symptoms or experienced mild symptoms. This aligns with previous reports suggesting that animals infected with SARS-CoV-2 generally have less severe illness compared to humans. However, it is essential to recognize the possibility of severe illness or death in animals, particularly those with underlying health conditions. Continuous surveillance of SARS-CoV-2 infections in companion animals is crucial for better understanding the virus's epidemiology in animals and developing effective strategies to protect both animal and human health.

Seasonal Patterns of Mpox Index Cases, Africa, 1970-2021.

Across 133 confirmed mpox zoonotic index cases reported during 1970-2021 in Africa, cases occurred year-round near the equator, where climate is consistent. However, in tropical regions of the northern hemisphere under a dry/wet season cycle, cases occurred seasonally. Our findings further support the seasonality of mpox zoonotic transmission risk.

Antimicrobial resistance in multistate outbreaks of nontyphoidal Salmonella infections linked to animal contact-United States, 2015-2018.

Animal contact is an established risk factor for nontyphoidal Salmonella infections and outbreaks. During 2015-2018, the U.S. Centers for Disease Control and Prevention (CDC) and other U.S. public health laboratories began implementing whole-genome sequencing (WGS) of Salmonella isolates. WGS was used to supplement the traditional methods of pulsed-field gel electrophoresis for isolate subtyping, outbreak detection, and antimicrobial susceptibility testing (AST) for the detection of resistance. We characterized the epidemiology and antimicrobial resistance (AMR) of multistate salmonellosis outbreaks linked to animal contact during this time period. An isolate was considered resistant if AST yielded a resistant (or intermediate, for ciprofloxacin) interpretation to any antimicrobial tested by the CDC or if WGS showed a resistance determinant in its genome for one of these agents. We identified 31 outbreaks linked to contact with poultry (n = 23), reptiles (n = 6), dairy calves (n = 1), and guinea pigs (n = 1). Of the 26 outbreaks with resistance data available, we identified antimicrobial resistance in at least one isolate from 20 outbreaks (77%). Of 1,309 isolates with resistance information, 247 (19%) were resistant to ≥1 antimicrobial, and 134 (10%) were multidrug-resistant to antimicrobials from ≥3 antimicrobial classes. The use of resistance data predicted from WGS increased the number of isolates with resistance information available fivefold compared with AST, and 28 of 43 total resistance patterns were identified exclusively by WGS; concordance was high (>99%) for resistance determined by AST and WGS. The use of predicted resistance from WGS enhanced the characterization of the resistance profiles of outbreaks linked to animal contact by providing resistance information for more isolates.

Antigenic mapping of the hemagglutinin of the H9 subtype influenza A viruses using sera from Japanese quail (Coturnix c. japonica).

IMPORTANCE: Determining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 hemagglutinin (HA) using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species.

Genomic epidemiology of third-generation cephalosporin-resistant Escherichia coli from Argentinian pig and dairy farms reveals animal-specific patterns of co-resistance and resistance mechanisms.

Control measures are being introduced globally to reduce the prevalence of antibiotic resistance (ABR) in bacteria on farms. However, little is known about the current prevalence and molecular ecology of ABR in bacterial species with the potential to be key opportunistic human pathogens, such as Escherichia coli, on South American farms. Working with 30 dairy cattle farms and 40 pig farms across two provinces in central-eastern Argentina, we report a comprehensive genomic analysis of third-generation cephalosporin-resistant (3GC-R) E. coli, which were recovered from 34.8% (cattle) and 47.8% (pigs) of samples from fecally contaminated sites. Phylogenetic analysis revealed substantial diversity suggestive of long-term horizontal and vertical transmission of 3GC-R mechanisms. CTX-M-15 and CTX-M-2 were more often produced by isolates from dairy farms, while CTX-M-8 and CMY-2 and co-carriage of amoxicillin/clavulanate resistance and florfenicol resistance were more common in isolates from pig farms. This suggests different selective pressures for antibiotic use in these two animal types. We identified the ß-lactamase gene blaROB, which has previously only been reported in the family Pasteurellaceae, in 3GC-R E. coli. blaROB was found alongside a novel florfenicol resistance gene, ydhC, also mobilized from a pig pathogen as part of a new composite transposon. As the first comprehensive genomic survey of 3GC-R E. coli in Argentina, these data set a baseline from which to measure the effects of interventions aimed at reducing on-farm ABR and provide an opportunity to investigate the zoonotic transmission of resistant bacteria in this region. IMPORTANCE: Little is known about the ecology of critically important antibiotic resistance among bacteria with the potential to be opportunistic human pathogens (e.g., Escherichia coli) on South American farms. By studying 70 pig and dairy cattle farms in central-eastern Argentina, we identified that third-generation cephalosporin resistance (3GC-R) in E. coli was mediated by mechanisms seen more often in certain species and that 3GC-R pig E. coli were more likely to be co-resistant to florfenicol and amoxicillin/clavulanate. This suggests that on-farm antibiotic usage is key to selecting the types of E. coli present on these farms. 3GC-R E. coli and 3GC-R plasmids were diverse, suggestive of long-term circulation in this region. We identified the de novo mobilization of the resistance gene blaROB from pig pathogens into E. coli on a novel mobile genetic element, which shows the importance of surveying poorly studied regions for antibiotic resistance that might impact human health.

Accumulation of resistance genes in Salmonella Typhimurium transmitted between poultry and dairy farms increases the risk to public health.

Salmonella Typhimurium is a zoonotic pathogen that poses a major threat to public health. This generalist serotype can be found in many hosts and the environment where varying selection pressures may result in the accumulation of antimicrobial resistance determinants. However, the transmission of this serotype between food-producing hosts, specifically between poultry layer flocks and nearby dairy herds, was never demonstrated. We investigated an outbreak at a dairy in Israel to determine the role of nearby poultry houses to be sources of infection. The 2-month outbreak resulted in a 47% mortality rate among 15 calves born in that period. Routine treatment of fluid therapy, a nonsteroidal anti-inflammatory, and cefquinome was ineffective, and control was achieved by the introduction of vaccination of dry cows against Salmonella (Bovivac S, MSD Animal Health) and a strict colostrum regime. Whole genome sequencing and antimicrobial sensitivity tests were performed on S. Typhimurium strains isolated from the dairy (n = 4) and strains recovered from poultry layer farms (n = 10). We identified acquired antimicrobial-resistant genes, including the blaCTX-M-55 gene, conferring resistance to extended-spectrum cephalosporins, which was exclusive to dairy isolates. Genetic similarity with less than five single nucleotide polymorphism differences between dairy and poultry strains suggested a transmission link. This investigation highlights the severe impact of S. Typhimurium on dairy farms and the transmission risk from nearby poultry farms. The accumulation of potentially transferable genes conferring resistance to critically important antimicrobials underscores the increased public health risk associated with S. Typhimurium circulation between animal hosts.IMPORTANCESalmonella Typhimurium is one of the major causes of food-borne illness globally. Infections may result in severe invasive disease, in which antimicrobial treatment is warranted. Therefore, the emergence of multi-drug-resistant strains poses a significant challenge to successful treatment and is considered one of the major threats to global health. S. Typhimurium can be found in a variety of animal hosts and environments; however, its transmission between food-producing animals, specifically poultry layers flocks and dairy herds, was never studied. Here, we demonstrate the transmission of the pathogen from poultry to a nearby dairy farm. Alarmingly, the multi-drug-resistant strains collected during the outbreak in the dairy had acquired resistance to extended-spectrum cephalosporins, antibiotics critically important in treating Salmonellosis in humans. The findings of the study emphasize the increased risk to public health posed by zoonotic pathogens' circulation between animal hosts.

Two episodes of bacteremia of zoonotic origin caused by different Streptococcus canis isolates in the same patient within a time span of 1 year.

Two episodes of bacteremia of cutaneous origin in a female patient were caused by two unrelated Streptococcus canis isolates within 1-year interval between the two infection episodes. The most likelihood transmission route in both episodes was a dog pet that habitually licked patient´s legs. Isolates were characterised by antimicrobial susceptibility test and whole genome sequencing. They belonged to sequence type (ST) 40 and 43, respectively. The ST40 isolate harboured antimicrobial resistance genes aadE, ermB and tetO, displaying resistance to erythromycin, clindamycin and tetracyclines, while ST43 isolate did not presented any known antimicrobial resistance determinant and was susceptible to all antibiotics tested. S. canis infections are rare in human; however, attention is needed for patients at risk with companion animals.

Genomic analysis of Brucella isolates from animals and humans, Türkiye, 2010 to 2020.

BackgroundBrucellosis is a bacterial zoonosis causing severe illness in humans and animals and leading to economic losses in the livestock production in Türkiye and other endemic countries.AimWe aimed at investigating genomic differences of Brucella isolates from animals and humans in Türkiye.MethodsWe used whole genome sequencing (WGS) to assess the genetic diversity of Brucella isolates from 41 provinces in Türkiye and compared with isolates from other countries. We applied allele-based typing and core genome single nucleotide polymorphism (cgSNP) determination.ResultsOf the 106 Turkish Brucella isolates included, 57 were B. abortus and 49 were B. melitensis. One B. melitensis and two B. abortus isolates were identified as vaccine strains. Most (n = 55) B. abortus isolates clustered in three major branches, with no spatial discernible pattern. Of the B. melitensis isolates, 48 were assigned to the Eastern Mediterranean lineage with no discernible patterns between host species, location and sampling date. The Turkish isolates clustered with isolates from neighbouring countries such as Greece and Syria, but some also with isolates from human patients in European countries, like Germany, Norway and Sweden, suggesting that the source may be travel-related.ConclusionSeveral B. melitensis and B. abortus lineages are circulating in Türkiye. To decrease the prevalence and prevent brucellosis in animals and humans, stricter control measures are needed, particularly in areas where humans and animals have close contact. Furthermore, illegal transportation of animals across borders should be more closely controlled and regulated.

Antigenic Distance between North American Swine and Human Seasonal H3N2 Influenza A Viruses as an Indication of Zoonotic Risk to Humans.

Human-to-swine transmission of influenza A virus (IAV) repeatedly occurs, leading to sustained transmission and increased diversity in swine; human seasonal H3N2 introductions occurred in the 1990s and 2010s and were maintained in North American swine. Swine H3N2 strains were subsequently associated with zoonotic infections, highlighting the need to understand the risk of endemic swine IAV to humans. We quantified antigenic distances between swine H3N2 and human seasonal vaccine strains from 1973 to 2014 using a panel of monovalent antisera raised in pigs in hemagglutination inhibition (HI) assays. Swine H3N2 lineages retained the closest antigenic similarity to human vaccine strains from the decade of incursion. Swine lineages from the 1990s were antigenically more similar to human vaccine strains of the mid-1990s but had substantial distance from recent human vaccine strains. In contrast, lineages from the 2010s were closer to human vaccine strains from 2011 and 2014 and the most antigenically distant from human vaccine strains prior to 2007. HI assays using ferret antisera demonstrated that swine lineages from the 1990s and 2010s had significant fold reductions compared to the homologous HI titer of the nearest pandemic preparedness candidate vaccine virus (CVV) or seasonal vaccine strain. The assessment of postinfection and postvaccination human serum cohorts demonstrated limited cross-reactivity to swine H3N2 from the 1990s, especially in older adults born before the 1970s. We identified swine strains to which humans are likely to lack population immunity or are not protected against by a current human seasonal vaccine or CVV to use in prioritizing future human CVV strain selection. IMPORTANCE Human H3N2 influenza A viruses spread to pigs in North America in the 1990s and more recently in the 2010s. These cross-species events led to sustained circulation and increased H3N2 diversity in pig populations. The evolution of H3N2 in swine led to a reduced similarity to human seasonal H3N2 and the vaccine strains used to protect human populations. We quantified the antigenic phenotypes and found that North American swine H3N2 lineages retained more antigenic similarity to historical human vaccine strains from the decade of incursion but had substantial differences compared to recent human vaccine strains. Additionally, pandemic preparedness vaccine strains demonstrated a loss of similarity to contemporary swine strains. Finally, human sera revealed that although these adults had antibodies against human H3N2 strains, many had limited immunity to swine H3N2, especially older adults born before 1970. Antigenic assessment of swine H3N2 provides critical information for pandemic preparedness and candidate vaccine development.

West Nile virus infections in Hungary: Epidemiological update and phylogenetic analysis of the Hungarian virus strains between 2015 and 2022.

Following the introduction of the West Nile virus (WNV) into Hungary in 2004, it has shortly become one of the most important human arbovirus infections, with a gradually increasing number of cases. The study aimed to summarize the current epidemiological situation in Hungary and sequence the WNV PCR-positive clinical specimens and virus isolates by next-generation whole genome sequencing (NGS) to obtain a detailed phylogenetic analysis of the circulating virus strains. Whole blood and urine samples from confirmed WNV-infected patients and WNV isolates were investigated by reverse transcription PCR assays. Genome sequencing was carried out by Sanger-method, followed by NGS on the Illumina MiSeq platform. Altogether 499 human infections were diagnosed between 2004 and 2022. A particularly remarkable increase in human WNV infections was observed in 2018, while the number of reported cases significantly decreased during the COVID-19 pandemic. Between 2015 and 2022, 15 WNV isolates, and 10 PCR-positive clinical specimens were investigated by NGS. Phylogenetic analysis revealed that the major European WNV lineage 2 clades, namely the Eastern European (or Russian) and the Central European (or Hungarian) clades, are presented in Hungary. Strains of the Balkan and other European clusters within the Central European clade are co-circulating in the country, following a characteristic geographical distribution. In Hungary, the presence and co-circulation of multiple lineage 2 WNV strains could be identified in the last few years. Therefore, in light of the 2018 WNV outbreak, sequence-based typing of the currently circulating strains could highly support outbreak investigations.

Description of eight human infections caused by Bacteroides pyogenes in a tertiary hospital of northern Spain.

Bacteroides pyogenes is a Gram-negative obligate anaerobe, saccharolytic, non-motile, non-pigment-producing and non-spore-forming rod. Reports of human infections caused by B. pyogenes are scarce, with approximately 30 cases documented in scientific literature. The aim of this study was to describe the clinical characteristics of 8 different patients and to study the in vitro antibiotic susceptibility of the strains, as well as to evaluate the in vivo activity of the prescribed treatments. We conducted a descriptive retrospective study by searching all B. pyogenes isolates from January 2010 to March 2023 at the Basurto University Hospital. This included all cases, both in monomicrobial or polymicrobial cultures. Among the eight patients, three experienced severe infections such as bacteremia and osteomyelitis. All the strains were susceptible to amoxicillin/clavulanic acid, piperacillin/tazobactam, imipenem, meropenem, clindamycin, metronidazole and moxifloxacin.

Tumor Necrosis Factor Alpha Contributes to Inflammatory Pathology in the Placenta during Brucella abortus Infection.

Research on Brucella pathogenesis has focused primarily on its ability to cause persistent intracellular infection of the mononuclear phagocyte system. At these sites, Brucella abortus evades innate immunity, which results in low-level inflammation and chronic infection of phagocytes. In contrast, the host response in the placenta during infection is characterized by severe inflammation and extensive extracellular replication of B. abortus. Despite the importance of reproductive disease caused by Brucella infection, our knowledge of the mechanisms involved in placental inflammation and abortion is limited. To understand the immune responses specifically driving placental pathology, we modeled placental B. abortus infection in pregnant mice. B. abortus infection caused an increase in the production of tumor necrosis factor alpha (TNF-α), specifically in the placenta. We found that placental expression levels of Tnfa and circulating TNF-α were dependent on the induction of endoplasmic reticulum stress and the B. abortus type IV secretion system (T4SS) effector protein VceC. Blockade of TNF-α reduced placental inflammation and improved fetal viability in mice. This work sheds light on a tissue-specific response of the placenta to B. abortus infection that may be important for bacterial transmission via abortion in the natural host species.

Francisella tularensis Exploits AMPK Activation to Harvest Host-Derived Nutrients Liberated from Host Lipolysis.

Francisella tularensis is a zoonotic, facultative intracellular bacterial pathogen that replicates in a variety of cell types during infection. Following entry into the cell and phagosome escape, the bacterium replicates rapidly in the cytoplasm. F. tularensis intracellular growth depends on the availability of metabolizable essential nutrients to support replication. However, the mechanism by which metabolizable nutrients become available to the bacterium in the intracellular environment is not fully understood. We found that F. tularensis-infected cells had significantly smaller and fewer lipid droplets than uninfected cells. Inhibition of triacylglycerol degradation significantly reduced bacterial growth, whereas inhibition of triacylglycerol formation did not reduce bacterial growth, suggesting that triacylglycerols sequestered within lipid droplets are important nutrient sources for F. tularensis. We found that F. tularensis-infected cells had increased activation of lipolysis and the upstream regulatory protein AMP protein kinase (AMPK). These data suggest that F. tularensis exploits AMPK activation and lipid metabolism to use host-derived nutrients. Finally, we found that AMPK activation is correlated with an increased bacterial burden, which suggests that it is a host-mediated response to nutrient starvation that results from increased bacterial replication. Altogether, we conclude that F. tularensis exploits AMPK activation to access nutrients sequestered in lipid droplets, specifically glycerol and fatty acids, to undergo efficient bacterial replication and cause successful infection.

SARS-CoV-2 Transmission from Human to Pet and Suspected Transmission from Pet to Human, Thailand.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been the cause of human pandemic infection since late 2019. SARS-CoV-2 infection in animals has also been reported both naturally and experimentally, rendering awareness about a potential source of infection for one health concern. Here, we describe an epidemiological investigation of SARS-CoV-2 infection in 639 cats and 224 dogs throughout multiple waves of COVID-19 outbreaks in Thailand. To indicate the potential source of infection, we performed SARS-CoV-2 genomic sequencing of samples obtained from pets and contacted humans, combined with in-depth interviews to support the epidemiological investigation. In the tested animals, SARS-CoV-2 RNA was present in 23 cases (19 cats and 4 dogs). Whole-genome sequencing of selected samples showed various SARS-CoV-2 variants of concern, which included the original European lineage (B.1), Alpha (B.1.1.7), Delta (B.1.617), and Omicron (BA.2). Among SARS-CoV-2-positive pets, 34.78% had evidence of contact with infected humans. Together with genomic analysis and an overlapping timeline, we revealed evidence of viral transmission from infected humans as the primary source, which spread to household cats via an undefined mode of transmission and most likely circulated between cohoused cats and caretakers within the weeks before the investigation. The SARS-CoV-2 surface glycoprotein (spike gene) obtained from caretakers of individual cats contained sequence signatures found in the sequences of infected cats, indicating possible exposure to the virus excreted by cats. Although pet-to-human transmission of SARS-CoV-2 is considered relatively rare, our study provides suspected episodes of human infection from animals that were initially infected through contact with infected humans.

Characterization of Swine Influenza A(H1N2) Variant, Alberta, Canada, 2020.

Influenza strains circulating among swine populations can cause outbreaks in humans. In October 2020, we detected a variant influenza A subtype H1N2 of swine origin in a person in Alberta, Canada. We initiated a public health, veterinary, and laboratory investigation to identify the source of the infection and determine whether it had spread. We identified the probable source as a local pig farm where a household contact of the index patient worked. Phylogenetic analysis revealed that the isolate closely resembled strains found at that farm in 2017. Retrospective and prospective surveillance using molecular testing did not identify any secondary cases among 1,532 persons tested in the surrounding area. Quick collaboration between human and veterinary public health practitioners in this case enabled a rapid response to a potential outbreak.

Septic Polyarthritis Caused by Streptobacillus moniliformis.

Streptobacillus moniliformis is a pleomorphic, fastidious gram-negative bacillus that colonizes rodent respiratory tracts and causes rat-bite fever in humans. Rat-bite fever is associated with septic arthritis, usually monoarticular or pauciarticular. We report a rare case of polyarticular septic arthritis caused by S. moniliformis; the disease was initially misdiagnosed as inflammatory arthritis.

Indoor and Outdoor Rodent Hosts of Orientia tsutsugamushi, Shandong Province, China.

During December 2012-July 2016, we tested small indoor and outdoor mammals in Qingdao, China, for Orientia tsutsugamushi infection. We found that outdoor Apodemus agrarius mice, Cricetulus barabensis hamsters, and Niviventer confucianus rats, as well as indoor Mus musculus mice, tested positive for O. tsutsugamushi by PCR.

Case-Control Study of Risk Factors for Acquired Hepatitis E Virus Infections in Blood Donors, United Kingdom, 2018-2019.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis in England. Substantial yearly increases of autochthonous infections were observed during 2003-2016 and again during 2017-2019. Previous studies associated acute HEV cases with consumption of processed pork products, we investigated risk factors for autochthonous HEV infections in the blood donor population in England. Study participants were 117 HEV RNA-positive blood donors and 564 HEV RNA-negative blood donors. No persons with positive results were vegetarian; 97.4% of persons with positive results reported eating pork products. Consuming bacon (OR 3.0, 95% CI 1.7-5.5; p<0.0001), cured pork meats (OR 3.5, 95% CI 2.2-5.4; p<0.0001), and pigs' liver (OR 2.9, 95% CI 1.0-8.3; p = 0.04) were significantly associated with HEV infection. Our findings confirm previous links to pork products and suggest that appropriate animal husbandry is essential to reduce the risk for HEV infection.

What a Difference a Gene Makes: Identification of Virulence Factors of Cowpox Virus.

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that causes spillover infections from its animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality compared to that caused by the mildly virulent laboratory strain Brighton Red (BR). The RatPox09 genome encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing the high virulence of RatPox09. To address this hypothesis, we employed several BR-RatPox09 chimeric viruses. Using Red-mediated mutagenesis, we generated BR-based knock-in mutants with single or multiple insertions of the respective RatPox09 genes. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses, and transcriptomic analyses confirmed that the expression profiles of the genes that were adjacent to the modified ones were unaltered. While the in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate, causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 (g7tGP) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching the rate for rats infected with RatPox09. The insertion of gCPXV0284, encoding the BTB (broad-complex, tramtrack, and bric-à-brac) domain protein D7L, also increased the virulence of BR, while the other two open reading frames failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model.IMPORTANCE With the cessation of vaccination against smallpox and its assumed cross-protectivity against other OPV infections, waning immunity could open up new niches for related poxviruses. Therefore, the identification of virulence mechanisms in CPXV is of general interest. Here, we aimed to identify virulence markers in an experimental rodent CPXV infection model using bacterial artificial chromosome (BAC)-based virus recombineering. We focused our work on the recent zoonotic CPXV isolate RatPox09, which is highly pathogenic in Wistar rats, unlike the avirulent BR reference strain. In several animal studies, we were able to identify a novel set of CPXV virulence genes. Two of the identified virulence genes, encoding a putative BTB/POZ protein (CPXVD7L) and a B22R-family protein (CPXV7tGP), respectively, have not yet been described to be involved in CPXV virulence. Our results also show that single genes can significantly affect virulence, thus facilitating adaptation to other hosts.

Immune Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase Virus Replication Fitness and Decrease Pandemic Potential.

H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T) and 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in field isolates in 2019. To investigate the potential threat of serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability, and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor-binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in the HA protein may pose a reduced pandemic risk but remain a heightened threat for poultry.IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry.