Nationwide genomic surveillance revealed the presence of viruses, bacteria, and antimicrobial resistance genes associated with animal and human health carried by domestic cats in China

doi:10.21203/rs.3.rs-3244858/v1

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Nationwide genomic surveillance revealed the presence of viruses, bacteria, and antimicrobial resistance genes associated with animal and human health carried by domestic cats in China

https://doi.org/10.21203/rs.3.rs-3244858/v1

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Background:

As a highly popular choice for a household pet worldwide, domestic cats may carry pathogens and antimicrobial resistance genes (ARGs) that pose severe risks to human health. However, our knowledge about pathogens and ARGs carried by cats remains limited. This large-scale genomic surveillance study is designed to identify viruses and bacteria as well as ARGs that may pose risks to animal and human health in the upper respiratory tracts of domestic cats in China.

Results:

Meta-transcriptome sequencing of 1454 oropharyngeal-nasal swabs collected from domestic cats in different living conditions in 22 Chinese provinces identified 371 viral species that can affect the health of animals and humans. A total of 24 viral species that had been known to cause infections in cats were detected. Among them, felis catus gammaherpesvirus 1, feline dependoparvovirus, feline foamy virus, fesavirus 4, puma lentivirus, and puma lentivirus-14 were detected in Chinese cats for the first time. Our study also identified the presence of nucleic acids from viruses associated with other animal species commonly found in human daily lives, including poultry, birds, pigs, cattle and other ruminants, minks, rabbits, and dogs. Notably, we identified many well-known viruses associated with public health concerns, including SARS-related coronavirus, monkeypox virus, and Zika virus (ZIKV). For the first time we found cats could be experimentally infected with and transmit ZIKV via mosquito bites, indicating a potential role of domestic cats in the maintenance and spread of the virus. We also identified a substantial number of bacterial species associated with public health, including the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) which showed a broad detection range. Additionally, a plentiful amount of ARGs, including those conferring resistance to the last resort antibiotics (carbapenems [bla_NDM, bla_OXA, bla_VIM], colistin [mcr], and high-level tigecycline [tetX3, tetX4, tetX5, tetX6]), were also detected broadly.

Conclusions:

This study indicate that domestic cats may act as a role in the worldwide emergence and increasing of zoonotic diseases and antimicrobial resistance. It is important to monitoring domestic cats within the One Health approach to control these critical global public health issues.

Domestic cats

genomic surveillance

virome

bacteriome

resistome

China

Emerging infectious diseases originating from animals pose a significant threat to global public health, resulting in a substantial burden of illness [1]. According to estimates, zoonoses are responsible for over one billion cases of human illness and millions human deaths worldwide each year [2, 3]. For the past two decades, the emergence and pandemic of infectious diseases with global impact, such as Severe Acute Respiratory Syndrome (SARS) in 2003, Zika virus disease in 2016, COVID-19 in 2019, and more recently, monkeypox, were all associated with pathogens originating from animals [4]. The World Organization for Animal Health (WOAH) has reported that, approximately 60% of the pathogens responsible for human diseases come from domestic animals or wildlife, while 75% of emerging infectious diseases in humans have an animal origin [5]. Therefore, the monitoring of zoonotic pathogens carried by animals, particularly those closely associated with human health, is essential for implementing One Health approaches. In addition to zoonotic infectious diseases, the urgent global public health crisis of antimicrobial resistance (AMR) also requires a One Health approach for control, due to the easy transmission of AMR between animals, environments, and humans [6, 7].

The domestic cat (Felis catus) is one of the most popular household pets worldwide, with its population having increased dramatically over the past 40 years. As of 2021, it is estimated that there are 600 million cats on earth in total, and 400 million of them live in Asia [8]. In China, there are 96 million cats being kept as house pets in 2021 and this number is expected to increase further [9]. Additionally, China also has a large population of stray cats. Due to the close relationship between cats and humans, it is important to monitor potential pathogens and harmful agents, such as antimicrobial resistance genes (ARGs), that may be carried by these animals and pose a risk to human health. For example, a recent case in Thailand reported a human contracting COVID-19 from a cat [10], highlighting the need for vigilance in this area.

Recently, whole genome sequencing (WGSs) combined with bioinformatic analysis has been utilized as a powerful tool to identify emerging pathogens that may pose a threat to public health [11]. One notable instance is the swift identification of SARS-CoV-2 as the pathogen responsible for the COVID-19 outbreak [12]. Moreover, WGSs has also been demonstrated to be useful in antimicrobial resistance surveillance due to its sensitivity and accuracy in detecting antimicrobial resistance genes or mutations [13]. In this study, we utilized the WGS approach to conduct a nationwide genomic surveillance on microorganisms (viruses and bacteria) and ARGs in upper respiratory tracts of domestic cats in China. Our research will not only shed light on the prevalence of pathogens that affect cat health, but also provide insights into the potential public health risks posed by this important household companion animal species.

Sample collection and virome profiling

From September 1, 2022 to October 30, 2022, we collected 1454 oropharyngeal-nasal swabs from domestic cats in catteries (509 samples), animal hospitals (449 samples), and stray bases (496 samples) across 22 provinces in China (Fig. 1A and Table S1). Samples were subsequently organized into 63 pools based on geographical regions and living conditions for meta-transcriptome sequencing, which led to the discovery of a vast number of viruses. Considering the close relationship of domestic cats to human lives, we mainly focused on viruses that are associated with specific groups of vertebrates, which include vertebrate-viruses that are closely related to virus families or genera known to infect humans, non-human primates, and domestic animals, such as cats, dogs, poultry and other birds, and livestock. Additionally, we also analyzed vector-borne viruses that are closely related to human lives, such as mosquitos, flies, mice, and ticks. Due to the frequent association of bat-borne viruses with emerging public health events, this group of viruses were also included for analysis.

According to the results of meta-transcriptome sequencing, a total of 371 viral species belonging to 50 known families and 12 unknown families that are associated with cat and human health as well as potential social and economic impact were characterized (Fig. 1B). Among them, viruses belonging to family Arenaviridae, Flaviviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Picobirnaviridae, Retroviridae, Sedoreoviridae, Rhabdoviridae, Adenoviridae, Papillomaviridae, Mitoviridae, Iridoviridae, Coronaviridae, Hepadnaviridae, Heterophyidae, Nairoviridae, Baculoviridae, Peribunyaviridae, Narnaviridae, and Caliciviridae were frequently detected and showed a relatively high abundance in cats living in diverse conditions and regions, while other viral families were only sporadically detected (Fig. 1B). The frequently detected viruses (> 85% sample pools) included cytomegalovirus, lentivirus, lymphocryptovirus, mammarenavirus, oryzopoxvirus, vesiculovirus, gammaretrovirus, orthoavulavirus, orthopicobirnavirus, alphapapillomavirus, mastadenovirus, orbivirus, rotavirus, flavivirus, simplexvirus, mitovirus, pestivirus, lymphocystivirus, orthohepadnavirus, and orthoreovirus (Fig. 2; Fig. S1).

Characterization of viruses associated with domestic cats and other animals

A total of 24 viral species that had been known to cause infections in cats were detected (Fig. 3A). Among them, feline calicivirus (FCV) was the most commonly detected virus with a broad geographical region of detection, followed by feline leukemia virus (FeLV), feline sarcoma virus (FSV), feline astrovirus (FeAstV), feline coronavirus (FCoV), rotavirus I, felid alphaherpesvirus 1 (FeHV-1), and feline infectious peritonitis virus (FIPV) in sequence (Fig. 3B). The detection of certain viruses in cats was found to be region-specific or dependent on the living conditions of the cats. For example, FeHV-1 was not detected in cats residing in southern and southwest China, and feline picobirnavirus (FPBV) was exclusively detected in cats from eastern China (Fig. 3). Feline immunodeficiency virus (FIV), FPBV, felis catus papillomavirus 3 (FcaPV-3), and fesavirus 4 were only detected in cats living in catteries, whereas feline bocaparvovirus 2 (FBPV-2), feline dependoparvovirus (FDPV), and norovirus GIV.2 were only detected in cats in animal hospitals (Fig. 3). Notably, felis catus gammaherpesvirus 1 (FcaGHV1), FDPV, feline foamy virus (FeFV), Fesavirus 4, puma lentivirus, and puma lentivirus-14 were detected in Chinese cats for the first time.

Our study also identified the presence of nucleic acids from viruses associated with other animal species commonly found in human daily lives, including poultry, birds, pigs, cattle and other ruminants, minks, rabbits, and dogs (Fig. 4, Fig. S2). The most frequently detected viruses for these animal species are avian avulavirus 1 (n = 62), porcine endogenous retrovirus (n = 60), mammalian orthoreovirus 5 (n = 54), classical swine fever virus (CSFV) (n = 48), porcine endogenous retrovirus A (n = 44), bluetongue virus (n = 61), bovine viral diarrhea virus (BVDV) (n = 54), and canine adenovirus (n = 41) (Fig. 4). Interestingly, African swine fever virus (ASFV) (n = 38), canine coronavirus (n = 12), and mink coronavirus 1 (n = 4) were detected (Fig. 4), along with several bat-borne viruses from eight known viral families (Fig. S3). Among them, viruses belonging to the family of Herpesviridae were frequently detected but their abundances were relatively lower than those from other families (Fig. S3). Additionally, five alphacoronavirus (α-CoV) were included in the detected bat-borne viruses.

Characterization of viruses associated with public health concerns

We also identified various 85 species may cause infections in humans and primates (Fig. 5), including SARS-related coronavirus, influenza A virus (IAV), and more strikingly, monkeypox virus, Zika virus (ZIKV), yellow fever virus (YFV), Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV), and various human-infecting viruses which are acquired mainly through blood, such as hepatitis C virus (HCV), and hepatitis E virus (HEV). Notably, SARS-related coronavirus, ZIKV, HBV, CCHFV, HCV, and HEV were detected in a broad geographic region (Fig. 5). For example, SARS-related coronavirus was detected in 35 sequencing pools from all seven regions in China, while ZIKV was detected in 55 sample pools from the same regions. However, IAV, YFV and monkeypox virus were detected sporadically (Fig. 5). IAV was only detected in16 sequencing pools from Northeast China (n = 2), Northern China (n = 2), Eastern China (n = 4), Southern China (n = 2), Northwest China (n = 2), and Southwest China (n = 4); while YFV was only detected in pools from Eastern (n = 2), Southern (n = 2), and Central (n = 1) China. Notably, monkeypox virus was detected in one pool each from Northeast and Northern China.

Domestic cats can be infected with and transmit ZIKV via mosquito bites

Given the frequent detection of ZIKV in various regions of China and the high risk of transmission of arthropod-borne viruses from animals to humans, we next investigated the potential of domestic cats to be infected with and transmit ZIKV. Aedes aegypti mosquitoes were fed on ZIKV-infected A129 mice in viremia period (Fig. S4A), resulting in an infection rate of approximately 70% in these mosquitos (Fig. S4B, C), while mosquitoes fed on mock-infected mice were used as a negative control. Subsequently, three 2-month-old domestic cats were exposed to mosquitoes that had fed on either ZIKV or mock-infected mice (Fig. 6A). Oral swabs were collected daily after mosquito-bites, while blood was collected every other day. At 5 days post infection (dpi), a relatively low abundance of viral RNA was detected in oral swabs from two out of three cats that were exposed to ZIKV-infected mosquitoes, while only one cat showed the presence of viral RNA in oral swabs at 6 dpi (Fig. 6B). However, all three cats showed obvious viremia at 4 and 6 dpi. This was determined by the detection of viral RNA in their blood (Fig. 6C) and the presence of infectious virus at 4 dpi (Fig. 6D). The above findings suggest that domestic cats can be infected with ZIKV through mosquito bites and harbor the virus in their blood.

To evaluate the potential transmission of ZIKV from domestic cats to other mammalian hosts, the infected cats in viremia period were subjected to biting by naive mosquitos (Fig. 6A). At 8 days post-bite, the mosquitos were fed on naive A129 mice for a second blood meal. The viral loads in both mosquitoes and mice were then determined. We found that about 15% of mosquitos were infected with ZIKV (Fig. 6E, F), and the viral RNA were detected in blood and various organs of mice (Fig. 6G, H). As a result, all mice died following a significant decrease of body weight (Fig. 6I, J). These findings reveal that the domestic cats infected with ZIKV is able to transmit the virus through mosquito bites.

To further verify whether the domestic cats can be infected with ZIKV in natural condition, the serum samples were collected from 18 adult cats living in stray bases in Ezhou, Hubei province, China. The neutralization antibodies against ZIKV were then detected using a plaque reduction neutralization test (PRNT), and the results showed that six cats had neutralization antibodies of ZIKV in their serum (Fig. S4D). This supports our conclusion that domestic cats may play a potential role in maintaining and spreading ZIKV.

Characterization of bacterial species with public health impact

To comprehensively understand the composition and prevalence of bacterial agents in domestic cats, the DNA samples were also subjected to metagenomic sequencing. The results revealed the presence of 18,381 bacterial species belonging to 2,926 genera (Table S2). The most abundant genera detected are Neisseria, Enterococcus, Klebsiella, Pseudomonas, Bordetella, Moraxella, Pasteurella, Streptococcus, Acinetobacter, and Staphylococcus (Fig. 7A). Notably, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) were commonly identified in the upper respiratory tracts of cats from various regions in China (Fig. 7B). In addition, several other species, including Pasteurella multocida, Streptococci, Staphylococci, Neisseria, Moraxella spp., and Bordetella bronchiseptica which frequently cause infections in humans via cat bites or scratches, were also commonly detected in upper respiratory tracts of cats from various regions with high abundances (Fig. 7B).

Characterization of antimicrobial resistance genes (ARGs)

The analysis of metagenomic sequencing data revealed the presence of large amounts of ARGs in the respiratory tracts of cats (Table S3 and Fig. S5). Multiple ARGs conferring resistance to World Health Organization (WHO) listed critically important agents for human medicine with highest priority, including 3^rd/4^th/5^th-generation cephalosporins (bla_CTX-M-series, bla_TEM-1/bla_TEM-131/bla_TEM-136, bla_SHV/bla_SHV-1, bla_PER-series, bla_VEB-series, bla_GES-13, bla_OXA-series), glycopepides (vanABCDEGHMNRSTUVXYZ), macrolides and ketolides (e.g., ermA, ermB, ermF), polymyxins (mcr-series), and quinolones (e.g., qnrA, qnrB, qnrS) (Fig. 8A). We next investigated the distribution of ARGs conferring resistance to last-resort antimicrobials, such as carbapenems, colistin, and tigecycline. Our analysis revealed the presence of three families of mobile carbapenem resistance genes (bla_NDM, bla_OXA, bla_VIM), one family of mobile colistin resistance genes (mcr), and one family of mobile genes mediating resistance to high-level tigecycline (tetX). These worrisome ARGs were widely detected in the respiratory tracts of cats from various regions of China (Fig. 8B). Cats from catteries and animal hospitals carried more ARGs than cats in stray bases, but there was no significant difference in the abundance of ARGs carried by cats from catteries and animal hospitals (Fig. 8C).

In this study, we conducted a nationwide survey to analyze the virome, bacteriome, and resistome composition in the upper respiratory tracts of domestic cats, which are commonly kept as household pets by humans. To our knowledge, this is the first large-scale genomic surveillance of viral and bacterial agents, as well as ARGs, in the upper respiratory tracts of domestic cats across China. By analyzing the pathobiome and resistome of 1454 samples, we were able to not only identify the distribution and prevalence of the main pathogens associated with the health of domestic cats in China, but also gain a comprehensive understanding about the microbial and antimicrobial resistant agents which are carried by this household species and may pose a threat to human health.

Although a large number of viral species were detected from the samples through meta-transcriptome sequencing, only a limited number of known viruses that had caused infections in cats were characterized. Among these feline viruses, FeLV and FCV were found to be highly prevalent among cats from various living conditions across different regions of China, suggesting a widespread distribution of these two viruses throughout the country. It has been known that FeLV infection is common in cats worldwide and is a leading cause of death [14]. However, there is a lack of vaccines against this viral species in China [15], which may explain the common frequency of detection of FeLV. Despite the availability of a vaccine against FCV in China [16], this viral species continues to be frequently detected and prevalent in a broad range of geographic regions. This might be attributed to a limited coverage of vaccination among the cat population in China. In addition to FeLV and FCV, many other viruses including FeAstV-2, FBPV-2, FCoV, FIV, FeKoV, CPPV-1, FPV, FPBV, FcaPV-3, Norovirus GIV2, FeTTV-1, FAV, and Rotavirus I, have been reported in China with lower detection rate than the aforementioned species [15, 17, 18]. However, FcaGHV1, FDPV, FeFV, Fesavirus 4, puma lentivirus, and puma lentivirus-14 have not been previously documented in China. The detection of these viral species may contribute to the complexity of feline viruses prevalent in China.

In addition to feline viruses, we also identified a large number of viruses that are associated with farmed animals, birds, and dog in the upper respiratory tracts of cats. It is possible that cats acquire these viruses or viral nucleic acids by consuming cat food that contains common ingredients such as chicken, pork, beef, and other food-producing animal products[19]. Dogs are also domestic species that are frequently kept as house pets. As a result, cats have frequent contact with dogs, which increases their risk of acquiring canine viruses or viral nucleic acids. Another notable finding for this study is that cats have been found to harbor a greater number of viral species associated with birds compared to other animals. This could be attributed to the innate hunting and killing behavior of cats towards birds [20], resulting in a higher likelihood of acquiring avian viruses. Although further studies are required to confirm whether cats play a role in maintaining and transmitting these viruses, it is advisable to prohibit raising cats and dogs in poultry and livestock farms in China, based on the genomic surveillance results of this study.

It is surprising that several viruses, such as Miniopterus bat coronavirus HKU8 [11], which are predominantly found in bats have been detected in some cats living in catteries and/or animal hospitals, as it is unlikely for cats living in these environments to have contact with bats. Further research is needed to determine whether these viruses can be sustained in cats. It is also surprising that many blood-borne viruses such as HBV and HCV were detected. It has been documented that China has one of the highest carrier-prevalence of HBV and HCV in the world [21-23], it indicates that cats may acquire HBV and HCV RNAs through close contact with human virus-carriers, although there have no reported cases of these two types of hepatitis viruses in cats. However, the detection of HEV in domestic cats has been documented recently [24, 25]. Therefore, it is less striking to find this species in cats. Additional research will be conducted to determine if Chinese cats can carry and transmit the three types of hepatitis viruses, through either the detection of antibodies or virus isolation.

It is of great notice that several mosquito-borne flaviviruses like ZIKV and YFV were detected in this study. Considering there are very few cases of ZIKV or YFV infection in China [26, 27], contact with virus-carrying mosquitos, either through mosquito bite or eating mosquitoes, might be a possible reason for cats acquiring these viruses, since cats are susceptible to mosquito bites, just like humans, and are also natural-born hunters, capable of catching and eating mosquitoes on occasions [20]. Our experimental results showed that cats can be infected with ZIKV through mosquito bites provides further evidence to support the hypothesis that cats may be a potential reservoir for the virus. This hypothesis is also supported by a previous study which has also shown an experimental infection of cats and dogs with West Nile virus, a flavivirus closely related to ZIKV, through both mosquito bites and predation [28]. Our study further demonstrated that the infected cats during viremia period could support the infection of mosquitoes, which in turn can lead to infection of mice by bite. However, the transmission efficiency of cats (~15% infection rate of mosquitos) is much lower than that of the A129 mouse model used in this experiment (~70% infection rate of mosquitos). This may be due to the lower levels of peak viremia in cats as compared to those in A129 mice. These findings indicate that domestic cats could potentially contribute to the transmission of ZIKV, albeit with a likely lower efficiency compared to the primate hosts. Similar principles could be applied to explain the detection of CCHFV in cats, as this tick-borne virus has also been identified in humans and ticks in China [29].

The detection of SARS-related coronavirus in cats from various regions is not beyond our expectations, as many studies have shown that cats are susceptible to SARS-CoV-2 infection during the early phase of COVID-19 pandemic [30-32]. Our sampling period (September 1~October 30, 2022) was near to the peak of COVID-19 in China in 2022 (December). During this period many people who had close contact with the cats for sampling, including cat owners, staff in catteries, animal hospitals, and stray bases, had been infected with SARS-CoV-2 during that time. This may have contributed to the cats acquiring the RNAs of SARS-related coronavirus. Monkeypox viral DNA was also detected from two sampling pools in northeast China and northern China, respectively, despite the absence of evidence of monkeypox virus currently affecting pets in China. However, it is important to note that monkeypox has been previously documented in China [33], and contacting with virus-carriers, including humans, might be a possible reason for cats acquiring monkeypox viral DNAs. Therefore, it is possible that cats may have acquired the virus through contacting with virus-carriers, including humans.

In addition to viruses, a large number of bacterial species which can cause severe infections in humans were detected in this study. Among these bacteria, the ESKAPE pathogens are known to be associated with multiple diseases in human, including pneumonia, bacteremia, blood-stream infection, and meningitis, and capture AMR determinants easily [34]. The healthcare industry has recognized infections caused by ESKAPE pathogens as a major problem, resulting in significant healthcare burdens for individuals and society worldwide [35, 36]. Our research suggests that the close contact between humans and domestic cats carrying ESKAPE pathogens in their upper respiratory tracts may increase the risk of pathogen transmission. In addition to ESKAPE pathogens, it is also important to consider the potential threat posed by other bacterial species to human health. For example, P. multocida has been increasingly reported to cause infection in humans via cat biting or scratching in recent years [37-39]. It has been documented that P. multocida could be isolated from 75% of the wounds induced by cat bites or scratches [40]. In this study, P. multocida was found to be commonly present in the upper respiratory tract of cats from all sampled regions in China, indicating a potential risk to human health.

Moreover, our study found that domestic cats may act as a reservoir of ARGs, since these genes were detected in abundance in their upper respiratory tracts. Most of these ARGs are linked to resistance against antibiotics that are commonly used in both human and veterinary medicine, even including many antimicrobials that are deemed critically important for human medicine by WHO (e.g., 3^rd/4^th/5^th-generation cephalosporins, glycopepides, polymyxins, quinolones, etc.) [41]. Mobile ARGs conferring resistance to carbapenems (bla_NDM, bla_VIM, bla_OXA), colistin (mcr), and high-level tigecycline (tet(X3), tet(X4), tet(X5), tet(X6)) have been commonly identified in various regions. These antibiotics are considered as the last options for treating infections caused by multidrug-resistant Gram-negative bacteria in humans, and are not approved for veterinary use in China [42]. One possible explanation for domestic cats bearing ARGs that confer resistance to last resort antibiotics and the other agents only used for humans is their close contact with human lives. This close contact may also facilitate the spread of these ARGs from cats to humans, thereby exacerbating the challenge of managing AMR. Therefore, in order to promote the One Health approach, it is important to take active measures to reduce AMR in cats and other animals [6, 7].

It has to be acknowledged that this study still has several limitations. To obtain a more comprehensive dataset, it would be beneficial to collect samples from a larger variety of cat groups including different breeds and from more various regions. Additionally, although cats from stray bases have been included in this study, most of these cats may have been kept in captivities for a period of time and may not accurately represent the pathobiome and resistome of stray cats. However, capturing stray cats in the field may present challenges due to their elusive nature. This study is also limited by a lack of data on isolating and/or culturing specific viral species from collected samples (e.g., ZIKV). Further research is currently underway to address this limitation.

Despite the above limitations, our study still provides comprehensive knowledge about the possible pathogens and ARGs carried by domestic cats from large geographic regions in China. This study not only systematically revealed the prevalence and distribution of known agents associated with cat and human health, but also identified the presence of several agents (FcaGHV1, FDPV, FeFV, Fesavirus 4, puma lentivirus, and puma lentivirus-14) that had not been previously reported in China. These data will contribute to the prevention and control of zoonotic diseases in China. Most notably, our research demonstrated for the first time that domestic cats may serve as a potential reservoir for ZIKV, thus increasing the risk of viral transmission from cats. We also identified a large number of microbes that can cause infections in humans and other mammals, as well as ARGs that confer resistance to commonly used antimicrobials in humans, in domestic cats from various regions in China. These findings suggest that domestic cats may play a role in the emergence and spread of zoonotic diseases and AMR. Therefore, it is important to monitoring domestic cats within the One Health approach to control these critical global public health issues.

Sample collection and treatment

Between September 1, 2022 and October 30, 2022, we collected 1454 oropharyngeal-nasal swabs from domestic cats in catteries (509 samples), animal hospitals (449 samples), and stray bases (496 samples) in 22 Chinese provinces (Fig. 1 and Table S1). This comprises 135 samples from northern China (39 samples from cats in catteries, 39 samples from cats in animal hospitals, 57 samples from cats in stray bases), 223 samples from northern China (96 samples from catteries, 75 samples from animal hospitals, 52 samples from stray bases), 285 samples from eastern China (111 samples from catteries, 58 samples from animal hospitals, 116 samples from stray bases), 273 samples from central China (96 samples from catteries, 77 samples from animal hospitals, 100 samples from stray bases), 160 samples from southern China (36 samples from catteries, 45 samples from animal hospitals, 79 samples from stray bases), 138 samples from northwest China (67 samples from catteries, 36 samples from animal hospitals, 35 samples from stray bases), and 240 samples from southwest China (64 samples from catteries, 119 samples from animal hospitals, 57 samples from stray bases). In each of the geographical regions, samples from cats in catteries, animal hospitals, and stray bases were organized into three pools, respectively. Therefore, the 1454 samples were finally organized into 63 pools. Sample collection as well as animal tests associated with this study was approved by the Animal Management and Ethics Committee of Huazhong Agricultural University. The reference ID number is 202303010001.

RNA/DNA extraction and Illumina sequencing

Total RNAs were extracted from each pool using a commercial RNAprep Pure Kit (TIANGEN, Beijing, China) while genomic DNAs were extracted using the cetyltrimethylammonium bromide protocol. After that, qualities and quantities of extracted RNAs/DNAs were assessed with 1% agarose gels electrophoresis system, NanoDrop2000 (Thermo Fisher Scientific, Waltham, US), and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, US), respectively. Sequence libraries for meta- transcriptome sequencing were prepared using an Illumina TruSeq™ RNA sample prep kit (Illumina, San Diego, China), and those for metagenome sequencing were prepared using a NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, Ipswich, US). Finally, barcoded libraries were paired end sequenced on Illumina Nova600 PE150 platform (Illumina, San Diego, China). This strategy yielded 8.82 Gb~18.63 Gb raw data for meta-transcriptome sequencing and 9.43 Gb~18.53 Gb raw data for metagenomic sequencing (Tables S4 and S5). Raw data from meta-transcriptome sequencing were de novo assembled using the Trinity program [43] with default settings; while those from metagenomic sequencing were assembled using MEGAHIT (version 1.1.2) [44] with default parameters.

Characterization of virome, bacteriome, resistome, and phylogenetic analysis

Viral species were determined by comparing assembled contigs against the NCBI non-redundant nucleotide (nt) and non-redundant protein (nr) databases using BLASTn [45] and Diamond BLASTx, respectively, with E value thresholds of 1 × 10^-6and 1 × 10^-4 to remove false positives. Sequences with RNA-dependent RNA polymerase (RdRp) in the annotation results of Conserved Domains Database (CDD) were screened out as candidate virus sequences. Finally, candidate viral sequences containing RdRp in the annotation results of CDD were compared with the RdRp sequences in GeneBank database. For viruses with large differences in sequences from known viruses, BLASTx was used to verify whether the sequences were from a novel virus.

To characterize bacterial species, clean reads were compared against NCBI nr database using Bowtie 2 (parameter: 95% identity) [46] and gene abundance in each of the samples was determined based on the alignment results with against the nr database (E-value ≤ 1e-5) by Diamond (version 0.8.35) [47]. The alignment results were then assessed based on the LCA algorithm and taxonomic lineage information was obtained for the top blast hit of each contig. ARGs were characterized by aligning unique genes against the SARG database (version 2.0) using ARGs-OAP v2.0 [48].

Experimental animal-infection and transmission assays

To explore whether domestic cats are susceptible to ZIKV and are capable to transmit the virus via mosquito bites, the following animal experiments were performed (Fig. 6A). Briefly, three 6-week-old IFNα/β receptor-deficient mice (A129 mice) were infected with ZIKV (strain H/PF/2013, GenBank accession no. KJ776791, 1000 PFU) through intraperitoneal injection. Another three mice received an injection of 100 µl Dulbecco's modified eagle medium (DMEM) were included as a mock-infection control. After three days of inoculation, viremia in murine blood was confirmed by detection of viral genome RNA using quantitative reverse transcription-PCR (qRT-PCR). Female Aedes aegypti mosquitoes were separated into netting-covered containers and starved for 12h before blood engorgement. The mosquitoes were allowed to feed on ZIKV- or mock-infected mice that were anaesthetised and placed on the top of containers for two days. After being anaesthetized on ice, the engorged mosquitoes were transferred to new containers and maintained under standard conditions for an additional three days to ensure viral multiplication. Afterwards, six 2-month-old SPF cats were divided into two groups and each group contained three cats. Each cat in the group was exposed to Aedes aegypti mosquitoes (n = 200), that had been fed on ZIKV- or mock-infected mice, for three days (2 h each day; day -2, -1, 0). Following the blood meal, the viral burden was determined in 36 out of 200 mosquitoes using qRT-PCR. From 0 to 10 days post infection (dpi), oral swabs were collected daily from each cat in the two groups, while blood was collected every other day. The viral RNA was detected using qRT-PCR, and the abundance of infectious virus in blood samples showing the peak viral RNA level (4 dpi) was determined by plaque assay. At 4, 5, and 6 dpi, each cat in different groups was exposed to naive Aedes aegypti mosquitoes (n = 50) to investigate the capability of mosquitoes acquiring ZIKV from cats. At 8 days post exposure, the mosquitos were then fed on naive A129 mice (n=3) for 2 days (2 h each day, day -1 and 0), and the viral load of part of mosquitoes was determined by qRT-PCR after blood meal (n=12 for mock-infection, n=27-35 for ZIKV-infection). The body weight and survival of mice were observed and recoded daily after mosquito-bites. The ZIKV burden in blood of mice was measured by qRT-PCR at 3 and 6 dpi. Additionally, the viral loads in various organs of the mice were assessed using qRT-PCR following the death of each mouse.

Quantitative reverse transcription-PCR (qRT-PCR)

RNAs from mosquitoes, cat blood, and mouse blood and tissues were extracted using the Trizol reagent protocol (Invitrogen, ThermoFisher, Waltham, MA, USA), and were reversed to cDNAs using a commercial ABscript II cDNA First Strand Synthesis Kit (ABclonal, Woburn, MA, USA). After that, cDNAs were used as template DNAs to detect nucleic acid copy numbers of ZIKV, with a pair of primers (F: 5’-CCGCTGCCCAACACAAG-3’; R: 5’-CCACTAACGTTCTTTTGCAGACAT-3’) and a synthesized probe (P: FAM-AGCCTACCTTGACAAGCARTCAGACACTCAA-TAMRA). PCR was performed in a 10-µl reaction system containing 5-µl of Entrans 2X qPCR Probe Master Mix V2 (ABclonal, Woburn, MA, USA), 10-µM of each forward and reverse primers, 10-µM of probe, 0.2-µl of 50× ROX dye (ThermoFisher, Waltham, MA, USA), 3-µM template cDNAs and 1.1-µM nucleic acid free water. The cycling conditions were 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, and 55 °C for 30 s.

Plaque assay and plaque reduction neutralization test (PRNT)

For plaque assay, samples were diluted and then inoculated to monolayers of VERO E6 cells. After incubation for 1 h at 37 °C, the unbound viral particles were removed, and cells were incubated with DMEM containing 2% fetal bovine serum and 1.5% sodium carboxymethyl cellulose (CMC) for 4 days at 37 °C in 5% CO2 incubator. Subsequently, cells were fixed with 10% formaldehyde and stained with crystal violet solution to count visible plaques. For PRNT, serial two-fold dilutions from origin to 1:64 of each serum sample was incubated with about 50 PFU of virus for 1 h at 37 °C. The virus-serum mixtures were added onto pre-formed VERO E6 cell monolayers and incubated for another 1 h at 37° C. The cell monolayer was then overlaid with CMC in cell culture medium and incubated for 4 days, followed by fixing and staining. Neutralization antibody titers were defined as the highest serum dilution that resulted in >50% reduction in the number of virus plaques (PRNT50).

Statistical analysis

Statistical analysis was performed using the two-way ANOVA test or the multiple-t-test strategy in GraphPad Prism 8.0 (GraphPad Software, San Diego, CA). Data represent mean ± standard deviations (SD) or mean ± standard error of mean (SEM). The significance level was set at a p value of <0.05 (*), a p value of <0.01 (**), or a p value of <0.001 (***).

AMR: antimicrobial resistance; ASFV: African swine fever virus; ARG: antimicrobial resistance genes; BVDV: bovine viral diarrhea virus; CCHFV: Crimean-Congo hemorrhagic fever orthonairovirus; CSFV: classical swine fever virus; α-CoV: alphacoronavirus; FCV: feline calicivirus; FeLV: feline leukemia virus; FSV: feline sarcoma virus; FeAstV: feline astrovirus; FCoV: feline coronavirus; FeHV-1: felid alphaherpesvirus 1; FIPV: feline infectious peritonitis virus; FPBV: feline picobirnavirus; FIV: feline immunodeficiency virus; FcaPV-3: felis catus papillomavirus 3; FBPV-2: feline bocaparvovirus 2; FDPV: feline dependoparvovirus; FcaGHV1: felis catus gammaherpesvirus 1; FeFV: feline foamy virus; IAV: influenza A virus; HBV: hepatitis B virus; HCV: hepatitis C virus; HEV: hepatitis E virus; SARS: Severe Acute Respiratory Syndrome; WGS: whole genome sequencing; WOAH: World Organization for Animal Health; YFV: yellow fever virus; ZIKV: Zika virus.

Ethics approval and consent to participate

Sample collection as well as animal tests associated with this study was approved by the Animal Management and Ethics Committee of Huazhong Agricultural University. The reference ID number is 202303010001.

Consent for publication

Not applicable

Availability of data and material

Raw data obtained from meta-transcriptome sequencing were deposited into NCBI Sequence Read Archive (SRA) database. The bioproject accession number is PRJNA998709. All other data is presented in the paper and available from the corresponding author on request.

Competing interests

The authors declare that they have no competing interests

Funding

This work was supported by National Key Research and Development Program of China (2022YFD1800105, 2022YFD1801500, 2021YFC2600204), National Natural Science Foundation of China (32022082, 31972721), Natural Science Foundation of Hubei Province (2021CFA056), and Fundamental Research Funds for the Central Universities (Project 2662023PY005).

Authors' contributions

Q.L., D.Z., and X.W. contributed equally to this article. Z.P., J.Y. and S.C designed the study. Q.L., D.Z., X.W., C.Z., S.H. and C.Q. performed the experiments. Q.L., D.Z., L.C. and W.W. collected and managed the samples. Z.P. and X.W. conducted the data analysis. Z.P. and J.Y. wrote the manuscript. J.Y., S.C. and H.C. supervised the study. J.Y. and S.C. acquired funds.

Acknowledgements

We thank our colleagues for their contribution to sample collection. The authors also acknowledge the Ministry of Natural Resources of the People’s Republic of China for providing standard maps for data presentation.

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No competing interests reported.

Additionalfile1.zip
Additional file 1: Fig. S1. A heatmap showing the distribution and detecting abundance of known viral genera in upper respiratory tracts of cats from different Chinese regions.
Fig. S2. A heatmap showing the distribution and detecting abundance of known viruses associated with poultry and the other birds, pigs, cattle and the other ruminants, minks, rabbits, and dogs.
Fig. S3. A heatmap showing the distribution and detecting abundance of known bat-borne viruses.
Fig. S4.Experimental infection and transmission assays of cats with ZIKV through mosquito bites. A. Detection of ZIKV viremia in the blood of A129 mice infected or mock-infected with ZIKV using qRT-PCR. Data are representative of 3 mice. Values are presented as Mean ± SEM. Each dot represents a mouse. B.Detection of ZIKV burden in Aedes aegypti mosquitoes using qRT-PCR after blood meal on A129 mice. Data are representative of 36 mosquitoes. Each dot represents a mosquito. The horizontal line represents the mean value of the results. C. Columns showing ratios and numbers of infected and uninfected mosquitoes from panel B. The numbers of infected and uninfected mosquitoes are shown at the bottom and top of each column, respectively. D.Detection of neutralizing antibodies against ZIKV in serum samples from cats in stray bases (n=18) using plaque reduction neutralization test (PRNT). The standard negative serum of cat was used as negative control (n=3). Data are representative of 3 or 18 serum samples. Values are presented as Mean ± SEM. Each dot represents a mosquito.
Fig. S5. A heatmap showing the distribution and detecting abundance of antimicrobial resistance genes (ARGs) in upper respiratory tracts of cats from different Chinese regions.
Additionalfile2.zip
Additional file 2: Table S1. Distribution of samples collected from cats for next-generation sequencing.
Table S2. Bacterial species detected in cats in different Chinese regions.
Table S3. Antimicrobial resistance genes detected in cats in different Chinese regions.
Table S4. Raw data and clean data obtained from different samples generated by meta-transcriptome sequencing.
Table S5. Raw data and clean data obtained from different samples generated by genome sequencing.

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Nationwide genomic surveillance revealed the presence of viruses, bacteria, and antimicrobial resistance genes associated with animal and human health carried by domestic cats in China

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