Rhinoviruses are small, single-stranded RNA viruses in the picornavirus family that are responsible for more than half of all upper respiratory tract infections. In addition to exacerbating asthma and chronic obstructive pulmonary disease, rhinoviruses have also been associated with acute respiratory hospitalizations among children (30). In a large prospective study of US pneumonias, rhinoviruses have been identified as the second most prevalent etiology of pneumonia in children after respiratory syncytial virus and the first most common etiology among adults (31). There are more than 150 unique types of rhinoviruses. Among the three genotypes (A, B, and C) types A and C are most often associated with increased morbidity and bacterial secondary infection. In animals, rhinovirus type C has been associated with morbidity in chimpanzees (32). With an array of unique serotypes no vaccines or approved antiviral therapies have been commercially produced; however, experiments have suggested that vaccines and antiviral therapy may be possible (33, 34).

Enterovirus D68 has caused sporadic respiratory disease outbreaks across Asia, Europe, and USA since 1960s; however, in 2014, a nationwide outbreak of D68 was associated with severe respiratory illness in USA, resulting in 14 deaths out of a known 1,150 cases (35). The CDC found 36% of all EVs tested during this outbreak were D68 and that patients with a history of asthma were found to be at a disproportionately increased risk of infection (36). One study of the 2014 outbreak found 59% of patients seen with EV-D68 in hospitals across Missouri, Illinois, and Colorado were admitted to intensive care units and 28% received ventilator support (35). In a study evaluating EVs in non-human primates, EV-D68 was detected as a recombinant zoonotic strain (37).

Influenza A viruses (IAVs) are segmented single-stranded RNA (ssRNA) viruses that infect a wide range of host species. At this time, wild aquatic birds are considered the primary natural reservoir host, from which novel viral lineages are periodically introduced into mammalian species, including humans, swine, equines, canines, and seals (1). Sixteen subtypes based on the primary antigen, hemagglutinin (H1 to H16), have been identified in wild birds, only two of which (H1 and H3) are currently established in mammals (humans, swine, canines, and horses). Human infections with avian H5 and H7 viruses have caused hundreds of deaths in Asia (2, 3), but so far, these viruses have not acquired the capacity for human-to-human transmission (4). However, H7 viruses were prevalent in horses until their replacement by H3 viruses, indicating that these viruses are able to establish infection cycles in mammals (5). Surveillance of IAVs is far greater in humans and birds than in other mammalian host species, and the origins of the 2009 pandemic influenza virus (H1N1pdm [pdm stands for pandemic]) in swine in Mexico underscore the threat presented by understudied mammalian populations in regions where viral diversity has been undetected for many years (6).

The canine respiratory tract contains both types of sialic acid receptors used by IAVs (α2,3- and α2,6-linked) (7). Dogs emerged as important IAV hosts in the 2000s, when IAV lineages in canines became established independently on two continents. H3N8 viruses were introduced from horses into canines in the United States (8), and H3N2 was introduced from birds into canines in Asia (9). Sustained transmission of the H3N8 lineage in U.S. canines occurs primarily in dog shelters with high rates of animal turnover (canine influenza virus H3N8 [CIV-H3N8]) (10). Equine H3N8 viruses were also transmitted to dogs in Australia and the United Kingdom, but transmission was not sustained (11, 12). The H3N2 lineage still circulates endemically in Asian dogs (CIV-H3N2), with reported cases in South Korea (13), Thailand (14), and multiple provinces in China (9, 15–17). A survey of canines in Guangdong Province in China reported that ~15% of farmed dogs and ~5% of pet dogs had antibodies in their serum to CIV-H3N2 (18). In 2015, CIV-H3N2 viruses were identified in U.S. dogs following a viral importation event from Asia (19). Multiple reassortant IAV genotypes have been identified in canines in Asia (see Fig. S1 in the supplemental material), including an H3N1 virus that acquired an N1 segment from a human H1N1pdm virus in South Korea (20). Reassortment between CIV-H3N2 and avian H5N1 viruses may also have occurred (21). Multiple avian virus segments have been identified in dogs, including H5N1, H6N1, and H9N2 (22–24), but the extent of onward transmission remains unknown. There are many outstanding questions concerning the genetic diversity, onward transmission, and spatial distribution of CIVs in Asia, but at the time of this study, only 54 full-length hemagglutinin (HA) sequences from CIVs in Asia were available in GenBank.

The expansion of the genetic diversity of CIVs since the 2000s has drawn comparisons to swine, which are considered key mammalian “mixing vessel” hosts. Swine sustain multiple IAV lineages acquired from human and avian hosts that exchange genome segments during coinfection via reassortment (25). The H1N1pdm virus provides a prime example of the mixing vessel capacity of pigs, as the pandemic virus genome contained segments from three different swine virus (IAV-S) lineages (26): (i) “classical” swine viruses (CswH1) that emerged during the 1918 H1N1 “Spanish flu” in North American pigs (27), (ii) the Eurasian avian-origin (EAswH1) lineage that originated in European swine in the 1970s (28), and (iii) “triple reassortant” swine H3N2 viruses (TRswH3) that were generated by reassortment events between avian, human, and swine viruses in North American swine in the mid-1990s (29). CswH1, EAswH1, and TRswH3 viruses all cocirculate in pigs in China, which has the world’s largest swine population (almost half a billion) and was initially considered a possible source of the H1N1pdm virus (26, 30). The identification of a progenitor virus of H1N1pdm in swine in Mexico was unexpected (6), but it does not diminish the pandemic risk presented by the large reservoir of IAV diversity in China’s swine, which only continues to expand following recent introductions of H1N1pdm viruses from humans (31, 32).

Opportunities for interspecies transmission of IAVs abound in southern China, where diverse species are often raised in proximity and intermingle at live-animal markets. In China’s Guangxi Zhuang Autonomous Region (Guangxi), dogs are kept as pets, roam as street dogs, and are raised for meat, providing opportunities for contact between canines and humans as well as other IAV host species at live-animal markets. Guangxi is located in southern China, bordering Guangdong Province and Vietnam (Fig. 1A), with a human population approaching 50 million. To further understand the genetic diversity of CIVs in this critically understudied region, we analyzed the presence of IAVs collected from dogs in Guangxi in 2013 to 2015, mainly from pet dogs presenting with respiratory symptoms at veterinary clinics. The complete genomes were sequenced for 16 isolated viruses (GenBank accession numbers MG254059 to MG254185). This resulted in the identification of five novel reassortant CIV genotypes that have not been previously described in canines, highlighting the capacity of dogs to serve ecologically as mixing vessels for reassortment between IAV lineages from multiple diverse host species.

From 2013 to 2015, 800 nasal swabs were collected from canines presenting primarily with respiratory symptoms at veterinary clinics in 11 of the 14 prefectures in Guangxi Zhuang Autonomous Region (Guangxi) (Fig. 1B). Of the 800 samples, 116 (14.5%) tested positive for influenza virus by PCR (Table 1). The influenza virus-positive animals were primarily pets (115/116) (see Table S1 in the supplemental material). Most were pure breeds, including Alaskan malamute (n = 16), bichon frise (n = 3), border collie (n = 3), chihuahua (n = 3), German shepherd (n = 7), golden retriever (n = 9), Siberian husky (n = 6), and poodle (n = 20). The majority of influenza virus-positive animals (~67%) were young dogs less than 1 year of age (median age of 5.5 months; range, 34 days to 9 years of age). The age profile of the influenza virus-positive animals did not differ significantly from influenza virus-negative animals (median age of 4 months; range, 2 days to 15 years). Twelve influenza virus-positive animals were rural Chinese dogs, including a 3-year-old female that died during treatment. It is unclear whether the death was caused by influenza. Approximately 70% (81/116) of the canines that tested positive presented with respiratory symptoms. Other animals presented with diarrhea, fever, vomiting, or injuries. Seven of the influenza virus-positive dogs were otherwise healthy. The first influenza virus-positive samples were identified in Liuzhou prefecture in April 2013 (Fig. 1C and Table S1). The first successfully isolated virus was collected from a 2-month-old female Samoyed presenting with respiratory symptoms in the Wuzhou district in October 2013 (Table 2).

Influenza virus-positive samples were identified in 9 of the 11 sampled prefectures and in each of the 3 years of sampling (2013 to 2015). Sampling was not conducted evenly across prefectures or across time (Fig. 1C and Table S2), so the percentage of positive samples in a location (Table 1) is likely to be biased and not appropriate for quantitative spatial-temporal comparisons. Sampling was conducted in multiple veterinary clinics in two prefectures (Nanning and Liuzhou), which explains the larger number of samples available from these locations (Fig. 1C). Sixteen viruses (2.0%) from seven prefectures spanning multiple regions of Guangxi were successfully isolated, and the whole genomes of the viruses were sequenced (Fig. 1B and Table 2). CIVs were isolated from five prefectures in the fall of 2013: Wuzhou (WZ), Hechi (HC), Fangchenggang (FCG), Qinzhou (QZ), and Liuzhou (LZ) (Fig. 2B). In 2014, CIVs were isolated in two additional prefectures: Chongzuo (CZ) and Nanning (NN). In 2015, CIVs were again isolated in Liuzhou and Nanning.

Since the first human infection by a novel avian influenza A H7N9 virus was reported in March 2013, a total of 458 confirmed cases with 177 deaths in China had been reported by December 2014.1 After a relatively silent period from July to October 2013, in which only four cases with one death were reported, the virus has reemerged since November 2013, resulting in the second outbreak in China.2 This novel influenza virus can bind to both avian (alpha 2,3-linked sialic acid) and human (alpha 2,6-linked sialic acid) receptors.3 It also contains human-adapted amino acid markers.4,5 These adaptations may explain why the virus can cause outbreaks in the human population.6 Patients infected with the H7N9 virus typically show symptoms such as fever, cough, opacities, and consolidation on chest radiography, and some severe cases can progress to acute respiratory distress syndrome (ARDS) and multiorgan failure.4 Although the lethality of H7N9 influenza is comparatively lower than that of highly pathogenic H5N1 viral infection, it is much higher than that of 2009 pandemic H1N1 influenza, reaching approximately 30%.7 Furthermore, high-pathogenicity markers for human-adapted influenza virus, such as an E637K amino acid substitution in the PB2 gene and Q226L in the haemagglutinin (HA) gene, have been identified in recent isolates of the H7N9 virus,4 suggesting that the virus might become more virulent in humans. Thus, the pandemic potential of lethal H7N9 influenza has raised public concern.

Although early treatments with oseltamivir and peramivir were effective,3,8,9 drug-resistant mutants of the virus were found soon after the patients received the anti-influenza therapy.10 Candidates of inactivated virus vaccines and virus-like particle (VLP) vaccines of H7N9 virus have been reported.11 However, the use of inactivated virus vaccines or VLP may not provide protection against mutated or reassorted influenza virus. In fact, a surveillance study has suggested that the novel influenza A H7N9 virus is a new reassortant of several influenza viruses, including H7, N9, and H9N2.6 Therefore, development of universal influenza vaccines is an attractive goal for scientists around the world. In this regard, the ectodomain of matrix protein 2 (M2e) of influenza viruses may be a promising target for the development of a universal influenza vaccine. This is because M2e is relatively conserved in different subtypes of influenza virus,12,13 and animal experiments have demonstrated that M2e vaccination can provide cross-protection against infection with different subtypes of influenza virus. H5N1-M2e-specific antibodies could react with different subtypes of influenza virus, such as H5N2, H9N2, H7N7, and H11N6.14 Various M2e-based vaccines have been developed, such as recombinant protein vaccines,15,16,17,18 plasmid DNA vaccines,19 and peptide vaccines.20,21 In our previous studies, we constructed a tetrameric H5N1-M2e vaccine candidate and demonstrated that it could provide cross-protection against lethal infections with different clades of H5N1 and 2009 pandemic H1N1 viruses.20,21 In this study, we extend our investigation to evaluate cross-protection of H5N1-M2e against lethal infection by the novel avian influenza A H7N9 virus in mice.

Six- to eight-week-old female BALB/c mice were provided by the Laboratory Animal Unit of the University of Hong Kong. Mice were maintained in cages and provided with sterilized food and water in the animal facility. The animal study was approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong.

Focussing on upper respiratory tract samples (nasal and tonsillar swabs), viral nucleic acids were detected in 31 of 214 diseased dogs (14.5%). Sixteen dogs tested positive for CRCoV (7.5%), 14 dogs for CPiV (6.5%) and one of these dogs additionally for CAV‐2‐specific nucleic acid (0.5%). One single dog tested positive for CDV‐specific nucleic acid (0.5%). In none of the obtained samples from the upper respiratory tract was CIV‐specific nucleic acid detected. Of those 31 positive dogs, 21 were privately owned (group A), and 10 kept in shelters (group B). They consisted of five puppies, 12 adolescent dogs and 14 adult dogs. Twenty‐seven of the 31 positive dogs (87.1%) showed acute onset of signs, three suffered from chronic disease (9.7%) and for one diseased dog this information was not available (Table 2).

Furthermore, upper respiratory tract samples from two dogs (4.0%) of the clinically healthy control group C tested positive for CRCoV‐specific nucleic acid (Table 2).

Nine dogs from group A (5.2%) and seven dogs out of group B (17.0%) tested positive for CRCoV in either nasal, tonsillar or both samples at one time. One of these dogs belonged to the subgroup of puppies; nine dogs were from the adolescent subgroup and six animals from the subgroup of adult dogs. With one exception, all these animals showed acute onset of CIRD (93.7%).

Fourteen diseased dogs (6.5%) tested positive for CPiV. From those, 11 belonged to group A and three to group B. They all harboured CPiV‐specific nucleic acid in sample material from the nose and one dog concurrently from the tonsils. Four of these dogs were classified as puppies; three dogs were from the adolescent subgroup and seven dogs were adults. Twelve out of these 14 animals showed acute onset of clinical signs (85.7%), one dog was chronically ill, and for another dog this information was not available. Seven dogs (50.0%) were regularly vaccinated‐including against CPiV.

In one of these 14 CPiV‐positive dogs, CAV‐specific nucleic acid was detected concurrently. This dog was privately owned (group A) and tested positive for CAV in both nasal and tonsillar swabs and CAV‐2 strain (Toronto) was confirmed by DNA sequencing. Belonging to the subgroup of adults this dog had been irregularly vaccinated and received its latest vaccine 45 days before sample collection. It presented with a several week history of clinical signs including severe coughing, nasal and ocular discharge, dyspnoea and fever. Apart from that case, in no other dog was viral nucleic acid of two or more different viruses detected. In addition, no other proband of the study tested positive for CAV.

One dog from group A tested positive for CDV‐specific nucleic acid in a sample retrieved from the tonsils. RNA sequencing enabled the identification of a CDV vaccine strain (Onderstepoort). The dog was an adult and presented with chronic respiratory disease but no other signs consistent with CDV infection. The vaccination status of this dog was unknown.

Additional information regarding all PCR‐positive dogs is summarised in Table 3.

All BALF samples collected from 31 chronically ill dogs revealed negative PCR results.

No conflicts of interest have been declared.

Analysis by multiplex PCR assays revealed that most dogs in both groups were positive for at least one CIRDV (96.9% in CAI and 94.7% in HAI groups). Among the six common CIRDVs, CIV and CRCoV were commonly found in both groups (CIV; 57.9% for CAI, 60.5% for HAI and CRCoV; 62.4% for CAI, 59.2% for HAI), while CAdV-2 was the least frequently detected (8.3% for CAI and 11.8% for HAI). The populations of other viruses in the CAI and HAI groups were CPIV, CaHV-1, and CDV, respectively. The CAI dogs had no statistical significance to the HAI dogs in term of virus detections (Table 1).

Multiple virus infections were detected at similar levels in both groups, at 81.2% (108/133) of CAI dogs and 78.9% (60/76) of HAI dogs, where double viral detections were the most frequently found (Table 2). The frequency of multiple virus detections in both groups decreased with increasing numbers of viruses, with no infection with all six viruses being found in either the CAI or HAI dogs. There was no significant difference between the CAI and HAI dogs in terms of multiple virus infections.

When the variable demographic factors and single or multiple CIRDV detections were analyzed, the main population was male and puppy in both groups. The common respiratory problems in both groups were nasal discharge, cough and depression. However, there was no association between sex, age, vaccination status and respiratory signs for single or multiple CIRDV detections (Table 3). The variable demographic factors were also compared with the clinical severity level, revealing that most CIRDV-infected puppies had a greater severity compared with the other age groups. There was a statistically significant association between the age of dogs and clinical severity level (P = 0.012), with the exception of sex, vaccination status, type of affected dog and number of different CIRDVs detected (Table 4). The respiratory score was compared with CIRD agents detected in both the CAI and HAI groups (Table 5). Most CIRDV-positive dogs expressed a respiratory score of 3 or 4 in both the CAI and HAI groups, which accounted for 60.2% (80/133) and 61.8% (47/76) respectively. Moreover, double infection with CIV and CRCoV was predominantly detected in both groups with a statistical association (P = 0.009, Table 5).

Influenza A virus has a wide range of hosts. Often the susceptibility of the species is dependent upon the characteristics of the virus and host. Numerous subtypes of influenza A viruses, including influenza A pandemic H1N1 2009 virus, have been shown to cross-species transmission. Since 2009, a novel influenza A virus (H1N1), now called A (H1N1) pdm09 influenza virus, has caused human influenza outbreaks in North America and a worldwide pandemic. To date, it has not only infected human, but also been reported interspecies transmission from humans to other animals, such as pigs, poultry, dogs.

Recently, the reports have shown that cats can also infected A (H1N1) pdm09 influenza virus. Due to frequent cohabitation and close contacts with humans and other animals, cats are uniquely positioned to serve as reservoirs for influenza virus infection both within a household and within the larger farm or rural environment in China. However, prevalence of A (H1N1) pdm09 influenza virus infection in cats in northeastern China is unknown. Therefore, the prevalence of A (H1N1) pdm09 influenza virus infections was performed among cats in northeastern China in this study.

A total of 1140 feline blood samples were collected from 56 different pet hospitals and four small animal shelters around northeastern China, from February 2012 to March 2013. The geographical and prevalent distribution of the samples has been concerned. Haerbin, Changchun and Shenyang were selected since they are the most densely populated area of commerce in northeastern China. Dalian was also included as it is the trade zone with large-scale breeding of poultry and pigs in northeastern China. The geographical location of serum samples of collection in northeastern China was displayed, please see the Figure 1. 660 blood samples from pet cats in hospitals and 480 blood samples from roaming cats were obtained. In each city, we selected the single largest small shelter. These serum samples were septed by centrifugation at 3,000 rpm for 15 min, and supernatants were transferred to a new eppendorf tubes and stored at-20°C until tested for antibodies against influenza A virus. Additionally, in order to have a timely data for pandemic (H1N1) 2009 prevalence in northeastern China, 115 blood samples were retrospectively analyzed from pet dogs and pet cats in Harbin in 2008. All samples were tested by hemagglutination inhibition (HI) and Neutralization (NT) assay, according to the recommended procedures as previously reported. HI titer ≥ 40 and NT titer ≥ 40 are considered as positive and indicate previous infection. Influenza virus used in this study was A/California/7/2009(H1N1pdm09) [pandemic (H1N1) 2009 virus]. We additionally studied the sera for HI antibodies against three other viruses: a human seasonal H1N1 influenza virus A/Brisbane/59/2007(H1N1) and A/canine/Guangdong/2/2011(H3N2), a recently circulating H3N2 canine influenza virus (CIV) in dogs in China. The comparison of categorical variables between cat samples was performed with chi-square test where appropriate. Statistical significance was defined as p < 0.05. The data was analyzed with SAS software, version 9.1.

A total of 1255 serum samples were examined by NT and HI for pandemic (H1N1) 2009 antibodies. The serological screening revealed 21% pandemic (H1N1) 2009 infection in cats in northeastern China based on NT. It also showed a higher prevalence rate of pandemic (H1N1) 2009 infection in pet cats (30.6%) than roaming cats (11%) based on NT (p = 0.0032, Table 1). The results from HI also showed a trend of difference in term of species of cats and it was statistically significant (P = 0.002). The prevalence of the infection also showed a geographical difference in roaming cats as prevalent in Harbin and Changchun (20.8% and 23.3%) and absent in Shenyang and Dalian (Table 1). In addition, the factors of the gender and age of the cats were also analyzed as contributors to pandemic (H1N1) 2009 prevalence. In the Table 2, while no influence of age (seropositive data not shown) was found on cats infection with pandemic (H1N1) 2009, genders associated with the pandemic (H1N1) 2009 seropositivity by both HI and NT assay was significantly (p < 0.05). In addition, a total of 115 serum samples collected in 2008 had no HI or NT antibodies against A/California/7/2009 (data not shown). To rule out non-specific cross-reactivity, 1140 serum samples were titrated against seasonal influenza viruses (H1N1). Only twenty-four samples had a HI titer of 1:40 against H1N1 (Table 3). Only ten of these forty seasonal influenza positive-samples were also HI and NT positive for A/California/7/2009(H1N1pdm09). A total of 111 (9.7%) sera were positive by HI assay against H3N2 CIV (Table 3).

Few seroprevalence studies on pandemic (H1N1) 2009 infections have been attempted in cats worldwide. The prevalence of this virus infection in cats in mainland China remains unknown. This is the first survey on the seroprevalence of pandemic (H1N1) 2009 infection in cats in northeastern China. Of all sera from cats in this study, 21% was identified as pandemic (H1N1) 2009 positive. In another conducting the seroprevalence of antibodies against (H1N1) pdm09 among cats in small cities of southern China was only 1.2% in 2011. Our increased antibody prevalence might be explained a number of ways. Perhaps cats were at a higher probability of infection in northeastern China, due to they exposures in dense populations of humans with high influenza A (H1N1) pdm09 attack rates. The difference might also be explained by the one year temporal difference between cats sampled in southern China in that the northeastern China cats had 1 more years to acquire influenza A (H1N1) pdm09 virus infection. Additionally, the prevalence of seropositive pandemic (H1N1) 2009 in male cats versus female cats suggests that the male cats may be more susceptible (P < 0.05) to the pandemic (H1N1) 2009 infections (Table 2). We hypothesize that relatively high A (H1N1) pdm09 transmission may have occurred between humans and cats during the period of virus infection in the human population. This hypothesis is supported by our observation that pet cats were more likely to have evidence of previous infection with A (H1N1) pdm09 that were roaming cats (30.6% vs11%, P = 0.0032) and also suggests a likely transmission between infected owners and their pets by close contact. Serological evidence of A (H1N1) pdm09 in domestic cats has been reported in the past. In a sero-survey conducted in Italy in 2009, a contrary low prevalence had been observed among dogs, while no cats were reported to have antibodies against A(H1N1)pdm09 in the screen. A similar high prevalence of 21.8% and 22.5% were recorded in a population of cats in the United States, but the study sample comprised animals with a history of respiratory disease. We hypothesized the sustained transmission of the influenza A (H1N1) pdm09 virus in the human population in our study area. In addition, it should be noted that 240 samples from the two small animal shelters in Harbin and Changchun had exposure to pandemic (H1N1) 2009 before sample collection. The higher prevalence of seropositive pandemic A (H1N1) pmd09 among Harbin and Changchun cats versus Shenyang and Dalian is unexplained.

Since cats may be exposed to different influenza virus subtypes, including human-avian and avian-origin influenza viruses, their potential role in the epidemiology of influenza virus should be further investigated. In summary, this study has observed a relatively high seroprevalence of pandemic (H1N1) 2009 in cats in northeastern China, similar seroprevalence studies should be conducted elsewhere. The studies showed that the prevalence for A (H1N1) pdm09 in human was correlated with age and population density. Preexisting antibody may have protected the very old from A (H1N1) pdm09 infection, while original antigenic sin and immunosenescence may have contributed to greater severity once infected. Compare with all serum samples collected in 2008 had no HI and NT antibodies against A/California/7/2009, these results reflect the pandemic (H1N1) 2009 had been spread in cats. Concerns of rapid spread in small animal shelters and household may be needed. These observations highlight the need for monitoring cats in pet hospitals and small animal shelters are necessary for us to understand what roles cats plan in the ecology of influenza A virus.

Infectious causes of respiratory disease are common in dogs; canine distemper virus, adenovirus 2, parainfluenza, influenza, herpesvirus, pneumovirus, respiratory coronavirus, Bordetella bronchiseptica, various Mycoplasma spp., and Streptococcus equi var. zooepidemicus are documented causes.1 Molecular diagnostic assays to detect viral and bacterial pathogens are available for these agents. In the United States, modified live vaccines (MLVx) for intranasal (IN) administration are currently available for adenovirus 2, B. bronchiseptica, and parainfluenza. These vaccines do not induce sterilizing immunity, and vaccinated dogs can still develop clinical signs of disease if exposed to virulent strains of the organisms.2 It is currently unknown if IN administration of MLVx against these agents results in positive molecular diagnostic assay results in dogs without previous vaccination. If transient positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays to predict disease caused by these agents in dogs would be decreased.

The purpose of this study was to determine the impact of administration of a single IN dose of a commercially available MLVx adenovirus 2, B. bronchiseptica, and parainfluenza containing vaccine,1 included as part of a facility standard initial vaccination series with a parenteral administration of MLVx containing adenovirus 2, canine distemper virus, and parvovirus, on the results of a commercially available polymerase chain reaction (PCR) panel that amplifies the RNA or DNA of the agents.2

The study was completed with Institutional Animal Care and Use approval. Beagle puppies housed at a commercial breeding facility were used.3 The puppies were housed in a closed facility without contact with other dogs and staff members followed facility barrier precautions over the course of the study. A sterile cotton swab was gently rubbed at the entrance to the external nares, and a second swab gently rubbed against the mucosa of the oropharynx in nonsedated puppies. The swabs were stored separately at 4°C in sterile plastic tubes and stored until shipped by overnight express on cold packs for performance of the molecular assays.2

A total of 12 puppies were screened twice as described, 1 week apart, and all were negative for nucleic acids of the target organisms. Eight puppies were randomly selected for the study and housed in a separate room at the breeding facility for the duration of the study. The puppies were approximately 9 weeks of age when samples were collected on Day 0 before the SQ administration of a MLVx containing adenovirus 2, canine distemper virus, and parvovirus4 and the IN administration of a MLVx1 containing adenovirus 2, B. bronchiseptica, and parainfluenza following manufacturer's instructions (approximately ½ mL per nares). Nasal and pharyngeal swabs were then collected on days 1, 2, 3, 4, 5, 6, 7, 10, 14, 17, 21, 24, and 28 for molecular analysis.2

Sneezing or coughing which have been associated with IN MLVx administration was not noted over the course of the study. Adverse effects associated with the collection of the nasal and oropharyngeal swabs were not noted. At the time the study was performed, the PCR panel utilized also included primers for canine distemper virus RNA; and none of the samples collected over the course of the study were positive. In contrast, nucleic acids of adenovirus 2, B. bronchiseptica, and parainfluenza were amplified from both sampling sites, from all 8 puppies, on multiple days after vaccine administration (Table 1). Because adenovirus 2 was administered in both vaccine types, source of that virus cannot be determined. Increasing numbers of positive samples after vaccination suggest local replication of the vaccinal strains. Decreasing numbers of positive samples over time suggest immune responses inhibiting organism replication. However, quantitative PCR assays normalized to total DNA/RNA on the swab would be needed to confirm or deny these hypotheses. The PCR laboratory adheres to standard operating procedures including use of positive and negative controls thus erroneous results are unlikely.

Agents considered most common for kennel cough syndrome include canine distemper virus, adenovirus 2, parainfluenza, and B. bronchiseptica. However, emerging pathogens include influenza, herpesvirus, respiratory coronavirus, pantropic coronavirus, pneumovirus, and others.1 All of these agents, as well as S. equi var. zooepidemicus and Mycoplasma spp., have been identified as causes of canine infectious respiratory disease. Determination of the agent is important for targeting treatment, particularly for dogs who fail to respond to standard treatment recommendations.2 In animal shelter environments, agent identification is critical for outbreak control and individual case management.3

Bacterial and viral shedding postvaccine administration complicates diagnostic testing and treatment. This is especially problematic in shelter environments as dogs are routinely vaccinated on intake. Viral shedding after vaccination has been detected in cats,4 people,5 cattle,6 pigs,7 and dogs.8 A vaccine strain of B. bronchiseptica was detected via nasal culture up to 4 weeks after IN vaccination of 2–week‐old puppies.9

Commercially available respiratory PCR panels are a relatively cost and time effective diagnostic method for identifying multiple respiratory pathogens. However, amplification of nucleic acids may inherently lead to inaccurate clinical diagnosis because small amounts can be amplified from some animals even though the agent may not be present in sufficient quantity to cause disease. In this study, nucleic acids of all 3 organisms contained in the IN vaccine were amplified from both sites on multiple days via PCR, although no clinical signs of respiratory disease were observed. Thus, interpretation of PCR panel results for diagnoses should include consideration of recent vaccination status and clinical signs of disease. Use of quantitative PCR and wild‐type sequence differences may be able to differentiate between vaccine and pathogenic agent shedding and may be used diagnostically in the future.

Real‐time reverse transcriptase PCR has been used to amplify canine distemper virus RNA in blood, urine, and conjunctival swabs after administration of SQ MLVx.10 In this study, the PCR panel did not amplify distemper virus RNA from nasal or pharyngeal swabs. Further studies are needed to determine whether the negative result is because this strain of vaccine virus does not reach the nasal or pharyngeal tissues or was present at levels below the detectable limit of the assay used.

The CIRDC, a common respiratory disease complex in dogs, is associated with environmental factors, individual host susceptibility and infectious pathogens, which are primarily viruses [1, 31, 32, 33]. The CIRDVs are commonly detected in dogs with respiratory problems and are endemic in poor conditioned dogs, overcrowded shelters, hospitals and pet grooming centers [2, 3, 8, 19, 34]. Currently, the prevalence and types of HAIs are not well established compared to CAIs. The epidemiology of CIRDV-infected dogs has been reporting over many years but with contrasting results [3, 8, 24]. In this study, we focused on the incidence of CIRDV detections in terms of CAI and HAI, which are supposed to be an important factor of CIRDC infections. The HAI-diseases, especially respiratory tract infections, have been considered as a risk for nosocomial transmission, but they have not been investigated in clinically-infected dogs [19, 34]. In this study, all six common CIRDVs were detected in both sample groups, suggesting that these viruses might either be present or disseminated in both environments. Moreover, we found that CRCoV and CIV had a higher detection rate in both the CAI and HAI dogs, while CPIV had a lower rate. This finding is in contrast to previous observations that found CPIV was the most commonly detected virus in CIRDC dogs [3, 7, 24]. Furthermore, it is surprising that 96.9% in CAI and 94.7% in HAI groups displayed at least one virus detection, which is unusual in previous observations for canine respiratory disease that revealed the low occurrence of CIRD viruses [4, 5, 7, 35], although this might be influenced by various factors, including the different geography, sample size, sample population, sampling protocols and also validated detection method.

The evidence of CDV, CPIV, CAdV-2, CaHV-1, and CIV circulation in Thailand has been documented previously [17, 18, 26, 36, 37], but not for CRCoV. This study, therefore, is the first report of CRCoV-infected dogs in Thailand. The most commonly detected viruses were CIV and CRCoV (>50%) in both the CAI and HAI groups, which is in contrast to previous reports in Asia and Europe [3, 7, 24, 39].

Meanwhile, CIRDVs were detected in both the CAI and HAI dogs without any significant difference in the frequency of occurrence among the two groups, potentially suggesting that these viruses are already circulating in both sample groups. This is in accordance with a recent study that reported that there was no significant difference in CPIV, CAdV-2 and CRCoV infection levels between dogs from private households and those from shelters or kennels [3, 40].

In the present study, 81.2% (108/133) of CAI and 78.9% (60/76) of HAI dogs had multiple CIRDV infections, supporting the complex condition of the disease, which is often manifested as a co-infection rather than a single pathogen [2, 3, 24, 26, 38]. We also observed that the majority of multiple CIRDV infections were co-detected with either CIV, CRCoV, or both. However, this observation does not allow interpretation as to whether they are primary and/or secondary pathogens. Infection with CIV and CRCoV usually induces mild clinical symptoms by interfering with the respiratory defense mechanism and so leads to super-infections with other pathogens [1, 41, 42]. Moreover, co-infection of CIV and CRCoV may be synergistic and lead to severe tracheobronchitis.

There were no significant differences in sex, age and vaccination status of the dogs between single and multiple virus detections. However, the vaccinated dogs had a lower proportion of both single and multiple detections compared with the unvaccinated dogs. Interestingly, there was a significant difference in the CIRD-affected age groups when compared with the clinical severity level. CIRD-affected puppies had a more severe clinical level compared with the dogs in the other age groups. This could reflect their increased host susceptibility, such as from a premature immune response and being unvaccinated. Further study with large-scale sample populations and meta-analysis of clinical information is warranted.

Significant association between CIRD agents and clinical respiratory scores was not observed in co-detection with CIV and CRCoV. This co-detection represented the highest proportion and was most often found with other CIRDVs. Thus, CIV and/or CRCoV could be either primary or secondary agents that are commonly found as co-infections with the other pathogens. However, bacterial infections could not be ruled out in this study. Many investigations have revealed that either viral or bacterial co-infection leads to an increased severity, but some studies have revealed no significant differences in the clinical severity between single and multiple infections [24, 39, 43, 44].

Since healthy or control dogs were not included in this study, then the prevalence of the CIRDV infections could not be assessed. Furthermore, this study used upper airway swabs as the sampling method for virus detection with PCR. It has been widely shown that healthy dogs may also have positive PCR results for CIRDVs in upper airway samples. Thus, a positive viral PCR in an upper airway sample is not necessarily proof of a symptomatic infection but could represent only exposure to the pathogen. Thus, the result of CIRDV detection by PCR from respiratory swabs can only imply that they represented the CIRD pathogens that might be associated with respiratory problem. Moreover, since we only focused on six viral pathogens associated with CIRDC, then the role of other pathogens, including known bacterial pathogens, such as Bordetella bronchoseptica and Mycoplasma sp., and other novel viruses, such as CnPnV, that might be implicated remains unknown.

FRZ and HYC designed the experiments. XY, DHZ carried out the test. PW,CGL and FRZ drafted the manuscript. All authors have read and approved the final manuscript.

Canine infectious respiratory disease (CIRD) is a multifactorial disease affecting dogs of all ages, which is typically induced by simultaneous viral and bacterial infections. Apart from well-known canine respiratory pathogens, such as canine adenovirus type 2, canine herpesvirus, canine distemper virus, and canine parainfluenza virus, novel viruses are being continuously associated with CIRD occurrence in dogs. These include canine influenza virus, canine respiratory coronavirus, canine pantropic coronavirus–[8], canine bocaviruses, and canine hepacivirus.

Pneumoviruses (family Paramyxoviridae, subfamily Pneumovirinae, genus Pneumovirus) are enveloped, single-strand negative-sense RNA viruses that are associated with respiratory disease in mammals and birds. Apart from the prototype species human respiratory syncytial virus (HRSV) and its ruminant relative bovine respiratory syncytial virus (BRSV), a murine pneumovirus (MPV), also known as pneumonia virus of mice, is included in the genus Pneumovirus

[11]. This virus, which is only distantly related to human and ruminant RSVs, is a natural rodent pathogen circulating among research and commercial rodent colonies.

Recently, a pneumovirus was associated to respiratory disease in canine breeding colonies in the United States–[14]. The virus, designated as canine pneumovirus (CnPnV), was found to be very closely related to MPV, displaying 95% nucleotide identity with the MPV prototype isolate J3666. Experimental infection of mice with the canine isolate demonstrated that CnPnV is able to replicate in the mouse lung tissue inducing pneumonia. Although the virus was discovered more than 4 years ago, to date there is no complete genomic sequence, which prevents a comprehensive comparative study with other members of the Pneumovirinae subfamily.

The aim of the present manuscript is to report the detection and molecular characterisation of this emerging virus in dogs with respiratory disease in Italy. The full-length genome of a prototype strain was determined and analysed in comparison with American strains and other pneumoviruses.

There are currently five other known canine parvovirus species belonging to two genera of the Parvoviridae family. Canine parvovirus 2 (CPV2) in the Carnivore protoparvovirus 1 species is a highly pathogenic virus that is closely related to feline parvovirus (FPV), the cause of feline panleukopenia, and can infect other carnivores such as coyotes, wolfs, raccoons and pumas. Canine bufavirus, a second protoparvovirus (in the species Carnivore protoparvovirus 2) was reported in 2018 in fecal and respiratory samples from both healthy and dogs with signs of respiratory illness. That same protoparvovirus was recently reported as a frequent component of juvenile cats fecal and respiratory samples. The canine minute virus (CnMV) in the Carnivore bocaparvovirus 1 species is less pathogenic than CPV2 but can cause diarrhea in young pups and is frequently found in the context of co-infections. Distantly related to CnMV, a second canine bocavirus in the Carnivore bocaparvovirus 2 species was sequenced in dogs with respiratory diseases. A third bocavirus was then characterized from the liver of a dog with severe hemorrhagic gastroenteritis.

Here, we describe the near complete genomes of two closely related cachaviruses, members of a new tentative species (Carnivore chapparvovirus 1) in a proposed genus Chapparvovirus, the third genera of viruses from the Parvoviridae family now reported in canine samples. The chapparvovirus was found in only two animals of the initial nine sampled. Many of the dogs in the outbreak analyzed were sampled more than 10 days after onset of clinical signs, increasing the possibility that they were no longer shedding viruses. Additionally, diarrhea is one of the top reasons for veterinary visits and some patients may have coincidentally presented with diarrhea from some other cause.

The two samples positive for CachaV-1 presented in the same week and were in the group of patients with the most severe clinical signs, requiring plasma transfusion and more aggressive supportive care. One of the two dogs, sampled at nine days after onset, died two days later. Because of the variable and often delayed feces sampling, it was therefore not possible to determine a clear disease association in this small group of diarrheic dogs (i.e., not all affected animals were shedding cachavirus).

A possible role for the cachavirus infection in canine diarrhea was further tested by comparing cachavirus DNA PCR detection in larger groups of healthy and diarrheic animals including a group of animals with bloody diarrhea. A statistically significant difference (p = 0.037) was seen when diarrhea samples from 2018 were compared to the feces from healthy animals collected the same year. When 2017 diarrheic samples were compared to e 2018 healthy samples, the p-value was 0.08. When 2017 and 2018 diarrhea samples were combined and compared to the healthy samples, the p-value was 0.05. The association of cachavirus with diarrhea is therefore borderline and the detection of viral DNA remains limited to ~4% of cases of diarrhea. The limited number of healthy samples available for PCR limited the statistical power of this analysis and a larger sample size will be required for further testing of disease association. The absence of detectable cachavirus DNA in 83 other cases of bloody diarrhea was unexpected given the similar signs that developed in the initial outbreak. Detection of viral DNA in feces may be related to timing of sample collection as shedding of the intestinal lining during hemorrhagic diarrhea may preclude viral replication and fecal shedding.

The detection of this virus in multiple fecal samples, the absence of prior cachavirus reports from tissues or fecal samples from other animals, and the confirmed vertebrate (murine) tropism of another chapparvovirus (mouse kidney parvovirus), support the tentative conclusion that cachavirus infects dogs. Given its relatively low viral load and only borderline association with diarrhea, this virus’ possible role in canine diarrhea or other diseases will require further epidemiological studies. Because viral nucleic acids in fecal samples may also originate from ingestion of contaminated food (rather than replication in gut tissues), the tropism of cachavirus for dogs will require further confirmation such as specific antibody detection, viral culture in canine cells, and/or evidence of replication in vivo such as RNA expression in enteric tissues of dogs shedding cachavirus DNA.

Rhonda LaFleur1, Tamara Davis1, Patrick Tuma1, Huchappa Jayappa1, Mike Francis2, Ian Tarpey2

1Merck Animal Health, Elkhorn, NE, USA, 2MSD Animal Health, Milton Keynes, UK

A study was conducted in dogs to evaluate the efficacy of an inactivated Canine Influenza Virus (CIV) H3N2 vaccine following experimental challenge with a virulent heterologous strain of CIV H3N2, isolated from the recent CIV H3N2 outbreak in the United States. Eleven dogs, 7–8 weeks of age, were vaccinated with 2 doses of an inactivated CIV H3N2 vaccine, 3 weeks apart, and 19 dogs were vaccinated with a placebo. Two weeks after the second vaccination, all dogs were challenged intranasally with virulent CIV H3N2 and then monitored daily for 10 days for clinical signs including fever, nasal discharge, sneezing, coughing, depression, and dyspnea. Nasal swabs were collected to evaluate viral shedding, and serum samples were collected at various time points to determine antibody titers. At necropsy, lungs were scored for consolidation. Following the booster vaccination, the placebo‐vaccinated control dogs remained seronegative (<10) to CIV H3N2, while 10 of the 11 vaccinated dogs developed an antibody titer to CIV H3N2 (GMT = <80; Range = <10 ‐ 320). Antibody titers in dogs from both treatment groups increased following challenge, but the increase was greater in the vaccinated dogs

(GMT = >1452; Range = 40 ‐ >10,240). Following challenge, 8 (42%) of the 19 placebo‐vaccinated control dogs were euthanized prior to the 10‐day post‐challenge observation period due to severe clinical signs, including difficulty breathing, depression, fever, and severe coughing with retching; whereas, none (0%) of the vaccinates had to be euthanized (P = 0.014). Clinical signs were evaluated based on a weighted scoring system. The mean clinical score for the placebo‐vaccinated control group was 24.9, compared to only 8.7 for the vaccinated group (P = 0.036). The placebo‐vaccinated control group shed CIV H3N2 virus for a mean of 1.9 days, compared to 1.4 days for the vaccinated group (P = 0.507). The median lung consolidation score for the placebo‐vaccinated control group was 7.4, compared to 0.0 for the vaccinated group (P = 0.026). Results of this study demonstrate that this inactivated CIV H3N2 vaccine significantly protects dogs against severe clinical disease and lung consolidation associated with a virulent CIV H3N2 infection.

Influenza A virus, a highly contagious pathogen, can infect both birds and mammals. It has undergone significant genetic variation to adapt to different hosts. Its interspecific transmission is achieved by the recombination or direct transfer of genetic material. The first case of dog infection with H3N8 canine influenza virus (CIV) was reported in the USA in 2004, followed by a report of CIV in South Korea, which subsequently demonstrated that CIV was able to transmit directly from dog to dog. Recently, the first case of H3N2 CIV infection was reported in Guangdong Province in 2010. Over recent years, infection with H3N2 CIV in dogs has developed from scattered cases to wide distribution across the country. Dogs have no natural immunity to this virus, thus a number of preventive and therapeutic measures against CIV have been attempted to control the prevalence of this virus. Among them, vaccination is an important method to prevent and control influenza virus infection. Current vaccine research against CIV has made some progress. In 2009, the U.S. Department of Agriculture (USDA) approved a list of vaccines against H3N8 CIV, which could effectively reduce viral shedding. In 2012, the patent for an H3N2 CIV vaccine in South Korea was also approved. Preventive vaccination is historically the primary measure to control influenza virus infection, but it has some limitations. For example, influenza vaccines may not be effective enough to prevent against divergent viral strains, or may be less immunogenic and effective in certain groups, such as the very young, the old, and the immunocompromised. Therefore, it is crucial to develop other measures to protect animals from infection/disease. For example, passive immunity by transferring a specific antibody to a recipient could protect animals from infection. Monoclonal antibodies (mAbs) can neutralize viruses, thus preventing virus attachment to, or fusion with, the host cell. Many studies have demonstrated that mAbs are an effective and preventive treatment against human-origin or avian-origin influenza virus infection. However, to date, there are no neutralizing mAbs available to prevent and control H3N2 CIV infection.

In this study, we identified seven mAbs against H3N2 CIV, and tested one of them, the D7 mAb, against three different H3N2 subtype virus strains in animal experiments. This is the first description of a neutralizing mAb against H3N2 CIV.

The study did not involve any animal experiment. Only sample collection from naturally infected dogs was carried out, consisting of a single nasal swab per dog. This was needed for the laboratory analyses and did not involve any suffering of the sampled animals.

Anne Cohen1, Jill Richardson2, Amy Glaser3, Edward Dubovi3, Nyssa Reine‐Salz2

1Chicago Veterinary Emergency and Specialty, Chicago, USA, 2Merck Animal Health, Madison, USA, 3Cornell University, Ithaca, USA

The H3N2 influenza virus is of avian origin and was first isolated from clinically ill dogs in China in 2006 and South Korea in 2007.1 Canine H3N2 influenza virus has been associated with severe respiratory signs and other clinical signs such as fever, reduced body weight, and interstitial pneumonia.1 This case report presents four confirmed, naturally infected clinical cases of Canine Influenza H3N2 in the United States. The cases presented were all pet dogs that had histories of recent exposure to other dogs in a social setting including dog parks and doggie day cares. None of the dogs originated from, or had traveled to, typical regions endemic for this viral disease. All dogs were presented at different stages of illness attributed to infectious respiratory disease.

One dog , a 1.5 year old Corgi, was evaluated for lethargy, anorexia, and loose stool for 2 days. He had also developed a cough on the day of presentation and had mild increases in respiratory rate. On physical examination, he had a fever with mildly increased respiratory sounds. The patient was re‐presented the next day for worsened signs of coughing, lethargy and hyporexia. Thoracic radiographs were performed which showed severe bilateral bronchopneumonia. Based on his poor response to supportive care, the owners elected humane euthanasia. Necropsy was performed at the New York State Veterinary Diagnostic Laboratory at Cornell University. Necropsy revealed Severe, acute, locally extensive necrohemorrhagic interstitial pneumonia with epithelial necrosis.

Another dog, a 8 year 10 month old Miniature Pincher, was evaluated after a 9 day history of coughing, wheezing, weakness, ataxia, hyporexia and one day of clear nasal discharge. On physical examination, the patient was weak, ˜7% dehydrated, had serous nasal discharge, muffled heart sounds, poor peripheral femoral pulses, and harsh bilateral thoracic sounds with crackles and wheezes. Thoracic radiographs were performed which showed right caudodorsal and left cranial bronchoalveolar bronchopneumonia. The patient experienced cardiopulmonary arrest and upon return of spontaneous circulation the owners elected humane euthanasia. Necropsy results were severe, acute, multifocal to coalescing necrohemorrhagic pneumonia with hyaline membrane formation and epithelial necrosis. Enteric lesions were also noted with moderate, diffuse, chronic lymphoplasmacytic enteritis with multifocal crypt necrosis.

Another dog, a 4 year old Greater Swiss Mountain dog, was referred for hospitalization due to right cranial and middle lung lobe consolidation, fever, dyspnea, gagging with phlegm production, exercise intolerance and hyporexia. On physical examination, he was quiet, febrile at 105.1 degrees Fahrenheit, had an increased respiratory rate of 60 breaths per minute with harsh bronchovesicular lung sounds. He became oxygen dependent with a pulse oximetry of 89–91%. Recheck thoracic radiographs were performed on the fourth day of hospitalization which showed a worsened alveolar pattern in the right and left cranial thorax. He was hospitalized for 7 days, making gradual improvements until discharge. H3N2 was confirmed via PCR at Cornell University.

The fourth case, a 10 year old terrier mix breed, was presented for evaluation of a productive cough for two days and one day of lethargy, anorexia and weakness. On physical examination, harsh lung sounds with mild increased respiratory rate was appreciated. The patient vomited a large volume of bile material after examination. A severe fever of 107.1 degrees Fahrenheit was appreciated with a respiratory rate of 48 breaths per minute and ˜5% dehydration. Thoracic radiographs were performed which showed a mild to moderate bronchointerstitial pattern most concentrated in the caudodorsal lung fields. The patient was hospitalized for 4 days and was eventually tapered off of oxygen and sent home on oral antibiotic therapy. A respiratory panel was sent to IDEXX Reference Laboratory. The patient was positive for an Influenza A Assay, negative for an H3N8 PCR and positive for H3N2 PCR.

All four of the dogs were diagnosed with Canine Influenza H3N2 through PCR, three through the New York State Veterinary Diagnostic Laboratory and one through Idexx Diagnostic Laboratory. In patients with characteristic clinical features, Canine Influenza H3N2 virus infection should still be considered as differential diagnosis.

It is clear that coronaviruses have high potential for emergence of novel variants with altered tropism and pathogenesis. In large part this can be explained by several factors: a combination of recombination and mutation within the viral genome and that many coronaviruses exist as quasi-species, the availability of natural reservoirs for the virus and the “modular” nature of the viral spike protein. Recent studies have highlighted the extraordinary complexity of canine coronavirus genomics. The increased likelihood of high-density housing for dogs increases the viral load of coronavirus in the population, and increases the likelihood of novel viruses emerging within dogs. It is now well-established that the feline coronavirus FIPV WSU-79-1146 arose following recombination with a canine coronavirus, and other recombinant canine-feline viruses have been identified. Increased co-housing of dogs with other species, particularly cats, also increases the chance of novel recombinant coronaviruses emerging across species.

To access the protective efficacy of mAb D7, we inoculated mice with three different H3N2 strains one day after treatment with mAb D7. Three days after the inoculation, all mice challenged with the three virus strains exhibited clinical signs of infection, including depression, decreased activity and huddling. Similar clinical signs were observed in irrelevant mAb IgG pretreated groups. However, similar to the PBS control group, mice in mAb D7 pretreated groups seemed to be energetic and had a good appetite during the infection.

In terms of body weight, mice challenged with JS/10 after treatment with D7 showed a similar increase in body weight compared with the PBS control group and there was no significant difference between the two groups (Figures 2A, B and C). At 14 dpi, mice treated with mAb D7 showed a body weight increase of nearly 30%. Although the body weights in the virus-infected group and irrelevant mAb IgG group both demonstrated an upward trend, the growth rate was slower than that in the mAb D7 group. The extent of the increase in body weight was significantly slower compared with that of the mAb D7 group at 10, 12 and 14 dpi (P < 0.05) (Figure 2A). In the group of mice infected with GD/12, the body weights of the mice in the three experimental groups all showed an upward trend, but the growth rate of the mice treated with mAb D7 was much higher than in the other two groups. In addition, the body weight changes of mice in the mAb D7 group at 10, 12 and 14 dpi were significantly different from those of the other two experimental groups (P < 0.05). The mice in the mAb D7 group showed weight gains of nearly 30%, which was not significantly different from the PBS control group (Figure 2B). However, after infection with SD/05, mice in the virus-infected group showed a slight decrease in body weight at 6 dpi and mice in the mAb IgG group displayed a slight decline at 8 dpi. By contrast, mice in the mAb D7 group continued to grow at 8 (P < 0.05), 12 (P < 0.01) and 14 dpi (P < 0.01). The growth rate in the mAb D7 group was significantly higher than that in the virus-infected group and mAb IgG group, but slightly lower than in the PBS control group at 14 dpi; mice body weight gain in the mAb group reached approximately 25% (Figure 2C).

Adenoviruses are common pathogens of vertebrates that were first discovered in human adenoids, and were soon identified as a cause of canine hepatitis. These icosahedral non-enveloped, double-stranded DNA viruses have genomes that range from 26 to 45 kbp, and have been demonstrated in all vertebrate classes. Most adenoviral species show quite restricted host specificity and tend to be associated with a typical pathology; for example, human adenovirus (HAdV) C causes respiratory disease and HAdV-D provokes conjunctivitis, whereas these two pathologies can also be the result of HAdV-B infection. In contrast, HAdV-F and HAdV-G produce gastroenteritis in most cases. Similarly to human adenoviruses, other adenoviruses that affect mammals (forming the Mastadenovirus genus) have been reported to cause respiratory, ocular and gastrointestinal pathologies, although some present as hepatitis or encephalitis as the chief manifestations.

In addition to their role in pathology, adenoviruses are very important vectors in the gene therapy of genetic disorders and cancer, as they can accommodate a large DNA cargo, exhibit tropisms for multiple organs and can be engineered to decrease virulence. Nonetheless, they still present toxicity problems, which has led to investigation of the potential of using animal adenoviruses as vectors for gene delivery to humans. In line with this, the identification of new animal adenoviruses, in addition to being interesting from an animal health perspective may be promising for gene therapy.

Sea lions are the only marine mammals in which adenoviruses have been recognized as pathogens. Adenovirus-like viral particles have been long since associated with hepatitis in stranded California sea lions (Zalophus californianus). More recently, a novel adenovirus (otarine adenovirus 1) was isolated from two stranded California sea lions with fatal hepatitis. This adenovirus caused an outbreak of fatal hepatitis and enteritis in three captive sea lions of different species: California sea lion (Zalophus californianus), South African fur seal (Arctocephalus pusillus) and South American sea lion (Otaria flavescens). In rare cases, adenoviruses have been isolated from gastrointestinal samples of other marine mammals, including a sei whale (Balaenoptera borealis), two bowhead whales (Balaena mysticetus) and a beluga whale (Delphinapterus leucas). Serological studies in Canadian fauna have also revealed antibodies against canine adenovirus 2 in 17% of the walruses (Odobenus rosmarus) examined. However, only in the case of sea lion hepatitis, has a clear association been established between the presence of virus and disease status. The partial sequence of the adenoviral DNA polymerase (pol) gene deposited in the Genbank (JN377908) was annotated as having been obtained from a harbour porpoise (Phocoena phocoena). However, there is no further information or referred publication available.

Here we identify a novel adenovirus in fecal samples of four captive bottlenose dolphins (Tursiops truncatus) which presented with self-limiting gastroenteritis. Gastric lesions, ulceration and parasitism are common in captive and free-ranging dolphins. However, reports of dolphin gastroenteritis are rare and the disease has never been associated with adenovirus. Pathological evidence for gastroenteritis has been reported in two necropsies of common dolphins from the Black Sea (Delphinus delphis ponticus) that showed evidence of systemic infection with Cetacean morbillivirus infection. Nevertheless, infections with this virus do not typically manifest as gastroenteritis and instead affect primarily the lungs and brain. Fatal gastroenteritis and toxic shock-like syndrome in dolphins has been attributed to enterotoxigenic Staphylococcus aureus. This animal concomitantly suffered brucellar osteomyelitis and was treated with antibiotics for nearly 1 year.

The present report describes several lines of evidence suggesting that adenovirus can be responsible for gastroenteritis in dolphins. Sequencing of PCR-amplified regions of adenoviral DNA (pol) and hexon genes revealed genetic closeness, but was not identical with the previously deposited sequences of sea lions and harbour porpoise adenoviruses. This suggests a close common ancestral origin of these viruses in marine mammals.

The factors regulating the course of the natural diseases caused by enteric CCoVs are not well understood. CCoVs are responsible for enteritis in dogs, and signs of infections may vary from mild to moderate, but they are more severe in young pups or in combination with other pathogens. Common signs include soft faeces or fluid diarrhoea, vomiting, dehydration, loss of appetite, and, occasionally, death. Dual infections by CCoV and canine parvovirus type 2 (CPV2) are especially severe when infections occur simultaneously, but CCoVs can also enhance the severity of a sequential CPV2 infection.

The natural route of transmission is faecal-oral, and virus in faeces is the major source of infection. In neonatal dogs, the virus appears to replicate primarily in the villus tips of the enterocytes of the small intestine causing a lytic infection followed by desquamation and shortening of the villi and resulting in diarrhoea 18–72 h post infection. Production of local IgAs restricts the spread of the virus within the intestine and arrests the progress of the infection. Therefore, infected dogs may shed virus for as long as 6 months after clinical signs have ceased [29, 38].

Recent extensive biomolecular analysis of faecal samples collected from infected dogs in Italy revealed that CCoVs infection is widespread and often characterized by the occurrence of both genotypes simultaneously [39, 40]. CCoVs type 1 and type 2 were found to be common in an Australian animal shelter with CCoV type 1 being prevalent. CCoVs have also been found in Western European dog populations. They have been detected in all European countries examined, and, except for the UK, the prevalence of CCoV type 1 was lower than for CCoV type 2. Reports of widespread CCoVs have come from Sweden and China. Soma et al. reported that CCoVs are also circulating in Japan, and the detection rate for dogs aged under 1 year was 66.3%, with a simultaneous detection rate of both types up to 40%.

These data raise several questions, and more indepth investigations into the pathobiology of CCoVs type 1 and type 2 are required. Therefore, failure to isolate CCoV type 1 in vitro hinders the acquisition of key information on the pathogenetic role of CCoV type 1 in dogs and prevents an authentic evaluation of the immunological characteristics of this new genotype.

Influenza A virus (IAV) has caused significant morbidity and mortality globally in humans, with an estimated 14 pandemics that have occurred since the 1500s.1 Wild aquatic birds are well known to be the natural reservoirs for IAV subtypes harbouring H1–H16 subtypes,2, 3, 4 with the exception of H17 and H18 subtypes that were recently discovered in bats.5, 6 The phylogenetic relationships of all IAV subtypes are displayed in Fig. 1. In addition to its natural reservoir species, influenza viruses infect a wide range of hosts including canids, equids, humans and swine.2 IAVs’ ability to generate novel gene constellations through reassortment between subtypes poses a risk for immune escape in these new hosts.7 Furthermore, IAV undergoes rapid genetic and antigenic evolution, which makes vaccination control difficult in humans and other domestic species.

In addition to human pandemics that have emerged from avian and swine hosts, there are also repeated spillover events from domesticated animals, primarily poultry and swine, that pose a significant threat to human health.8, 9, 10, 11, 12, 13, 14 Direct transmission of IAV from a wild avian source to humans is rare, as there has only been a single report of laboratory‐confirmed human infection with H5N1 contracted through close contact with dead and infected wild swan in Azerbaijan.15 However, there is serological evidence of H5N1 infection among Alaskan hunters who handled dead wild avian species,16 indicating that exposure to IAVs from wild birds through close contact can potentially cause infection. More notably, viral genes that are similar to the 1918‐like H1N1 avian virus were recently detected in the influenza gene pools of wild birds, raising the potential for the re‐emergence of a 1918‐like pandemic virus.17 Furthermore, due to increasing human encroachment of wildlife habitats, the potential of a wild‐source threat becomes more relevant, as is seen with the emergence of other pathogens such as human immunodeficiency virus (HIV), severe acute respiratory syndrome coronavirus and the more recent Zaire‐variant Ebola virus in Western Africa.18, 19, 20, 21

In this review, we discuss the current knowledge of ecological and molecular determinants responsible for interspecies transmission of IAV, with specific focus on avian‐derived influenza subtypes involved in zoonotic and epizootic transmission to other hosts (see Fig. 2).

In summary, we report herein self-limiting gastrointestinal disease in bottlenose dolphins, which appears to be due to a hitherto unknown adenovirus that is genetically close to the adenoviruses found previously in marine mammals, such as sea lions. Adenoviral causality is supported by the exclusive detection in the four diseased animals, concomitantly with the disease, of adenovirus in feces and of antibodies for canine adenoviruses 1 and 2 in the serum. While adenoviral infection of sea lions causes hepatitis and death, the dolphins infected with putative tursiops adenovirus 1 suffered self-limiting disease, with no signs of hepatitis. In addition, the dolphins showed no apparent respiratory or ocular pathologies, which are frequent in adenoviral infections of many other species. Thus this adenovirus may not show liver, lung or eye tropism. Full adenoviral genome sequencing might help predict tropism, since certain genetic elements have been associated with certain tropisms. The full viral sequence might also be necessary to more broadly confirm our present inference, based on the limited phylogenetic analysis of partial pol and hexon gene sequences (Figure 2), and that this adenovirus is more closely related with adenoviruses of other marine mammals than with those of other taxonomic groups. This close relation suggests that a branch of the adenoviral tree evolved when marine mammals became adenoviral hosts. It remains to be elucidated whether adenoviruses represent a serious threat to dolphins. In any case, the present findings highlight the need to consider this adenovirus a causal agent of dolphin gastroenteritis, which should be taken into account in the differential diagnosis of this condition, at least for captive dolphins.