A variety of inactivated and modified-live virus vaccines are commercially available and designed to prevent infection with CCoV. Current vaccines are safe, but provide only incomplete protection—in that they reduce, but do not eliminate, CCoV replication in the intestinal tract. As these vaccines are likely based on the classical CCoV-IIa viruses, protection against CCoV-1 strains with these vaccines is unlikely, and protection against the variant type II strains is uncertain. Treatment of CCoV-induced gastroenteritis is mainly by supportive care, including good maintenance of fluid and electrolytes. There are no available anti-viral drugs for treatment of CCoV infections.

No additional information is available for this paper.

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.

During the last two decades, scientists have grown increasingly aware that viruses are emerging from the human–animal interface. In order to combat this increasingly complex problem, the One Health approach or initiative has been proposed as a way of working across disciplines to incorporate human, animal, and environmental health. Of particular concern are emerging respiratory virus infections; in a recent seminar given by the National Institute of Health on emerging and re-emerging pathogens, nearly 18% were respiratory viruses (1). Among the recently emerged respiratory pathogens contributing to the high burden of respiratory tract infection-related morbidity and mortality, displayed graphically in Figure 1, are influenza viruses, coronaviruses, enteroviruses (EVs), and adenoviruses (Ads). In this report, we summarize the emerging threat characteristics of these four groups of viruses.

To control potential new influenza pandemics, the best measure may be to develop a universal influenza vaccine because no existing vaccines can provide effective cross-protection against a mutated or reassorted novel influenza virus. Current efforts to develop universal vaccines include the development of M2e-based vaccines, HA stem vaccines 29 and M1 VLPs.30 M2e-based vaccine candidates can provide cross-protection against infections of different subtypes of influenza virus.20,21,31 In our previous studies, we showed that an H5N1-M2e-based tetrameric peptide vaccine exhibited promising protection against lethal challenge with different clades of H5N1 virus and other subtypes of influenza virus.20,21 In this study, we further demonstrated that this tetrameric peptide vaccine could also offer potent protection against infection with a novel H7N9 influenza virus, which caused a first outbreak from March to June 2013 and a second outbreak from November 2013 to December 2014 in China.

Our results showed that the H5N1-M2e tetrameric peptide vaccine, together with either FA or SAS, induced a high antibody response that was able to effectively cross-react with H7N9-M2e (Figure 2). The vaccinations thus protected the mice from a lethal challenge with the H7N9 virus (Figure 3). The survival rate of H5N1-M2e-vaccinated mice against a H7N9 lethal challenge was 80%, which was the same as that of H5N1-M2e-vaccinated mice against a H5N1 lethal challenge.20 Consistently, viral load and lung damage in H5N1-M2e-vaccinated mice were much lower and less severe than that in adjuvant- or PBS-injected mice (Figure 4). Although T-cell responses induced by an M2e-based vaccine have been reported previously, the protection by the M2e vaccination was mainly attributed to the induced antibodies but not the T cells.19 Similarly, our results showed that the cross-protection of the H5N1-M2e vaccination was directly related to the levels of cross-reactive antibodies toward the M2e of challenge viruses. Unlike vaccinations with inactivated viruses, HA-subunit vaccines and VLP vaccines, which may provide complete protection against infection by homogenous viruses but poor cross-protection against infection by heterogeneous viruses, our results illustrate that the H5N1-M2e tetrameric peptide vaccine can provide satisfactory cross-protection against infection with a novel influenza virus that has newly emerged in humans.

Amino acid mutations at positions 10 and 11 of M2e reduce the monoclonal antibody binding affinity,32 whereas virus mutants at M2e position 10 can escape the protection of M2e antibodies.33 Moreover, M2e peptide variants at positions 10, 14, and 16 significantly reduce reactive antibody-binding titer.19 These findings indicate that although M2e is relatively conserved among different subtypes of influenza virus, amino acid mutations at certain positions could indeed affect the efficacy of M2e vaccination. Notably, there are five amino acid differences at positions 13, 14, 18, 21, and 24 between H5N1-M2e and H7N9-M2e, which accounted for approximately 21% (5/24) of the total amino acids of M2e (Figure 1). However, these amino acid variations did not abolish the efficacy of the vaccination. The protection against lethal challenge by H7N9 virus (Figures 3 and 4) was not affected, despite the fact that the amino acid variations between H5N1-M2e and H7N9-M2e included a mutation at position 14, which has been reported to affect the antibody cross-reaction.19 To examine how the amino acid variations may affect the structure of the M2e peptide, the 3D structures of M2e-A/Vietnam/1194/04 (H5N1), M2e-A/Hong Kong/156/97 (H5N1), M2e-A/Anhui/01/13 (H7N9), and M2e-A/Beijing/501/09 (H1N1) were analyzed using the online software PEP-FOLD (Figure 5). The 3D structures of M2e-A/Vietnam/1194/04 (H5N1) and M2e-A/Anhui/01/13 (H7N9) showed that the middle regions were similarly folded as an “open” structure, although there were five amino acid differences between these two M2e sequences (Figure 1). Although there was only one amino acid difference between M2e-A/Vietnam/1194/04 (H5N1) and M2e-A/Beijing/501/09 (H1N1), the 3D structure of M2e-A/Beijing/501/09 (H1N1) showed a relatively more “open” structure (Figure 5). However, M2e-A/Hong Kong/156/97 (H5N1), which contained amino acid variations at positions 10, 14, and 16 (Figure 1) and had low cross-reactivity with the H5N1-M2e-induced antibody (Figure 2C), had the same region folded as a hairpin structure. According to the 3D prediction model, the 12th amino acid, arginine, and the 15th amino acid, tryptophan, in the structure of M2e-A/Hong Kong/156/97 (H5N1) are bulky and are located at the top of the hairpin, which may provide steric hindrance that blocks the access to the lower region of the structure. This structure may limit the antibody-binding ability and the antigen processing by the immune system. By contrast, the 12th-position arginine and the 15th-position tryptophan of the other three M2e structures orientate in such a way that they would not block the lower access. These observations may explain why the H5N1-M2e vaccine-induced antibody could still react with M2e that contained variations at positions 13, 14, 18, 21, and 24, but it could not react with M2e containing amino acid variations at positions 10, 14, and 16, because their 3D structure were not compatible.

A slight protection (10% survival) was observed in mice vaccinated with FA alone (Figure 3). A similar phenomenon was also reported in other studies, in which vaccination with FA alone provided a slight protection (10% survival) against lethal challenge by enterovirus 71 and group A Streptococcus, respectively.34,35 A possible explanation is that the adjuvant alone may induce innate immune responses that can provide a certain level of protection against viral infections.36 Nevertheless, the survival rate of the mice vaccinated with H5N1-M2e plus FA was significantly higher than that of the mice vaccinated with FA alone (P < 0.01), indicating that it was H5N1-M2e but not the adjuvant that provided the key protection against lethal challenge with the H7N9 virus.

In summary, this study has illustrated that H5N1-M2e may provide potent cross-protection against lethal challenge from a novel avian influenza A H7N9 virus, even though approximately 21% amino acids were different existed between H5N1-M2e and H7N9-M2e. These results suggest that the M2e tetrameric peptide may provide broad spectrum of cross-protection against infections by heterogeneous influenza viruses. The M2e tetrameric peptide may be a promising candidate for the development of a universal vaccine. To improve the vaccine's protection, the M2e vaccine may be used together with inactivated virus, HA-subunit and/or other types of vaccines. Complete protection has been reported in the combined use of an M2e-based vaccine and inactivated virus vaccine.37

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.

Outbreaks of Ad in the general population have been characterized by infection due to novel viruses such as Ad7h, Ad7d2, Ad14a, and Ad3 variants. These novel viruses are sometimes associated with high attack rates and a high prevalence of pneumonia. Severe mortality is also prevalent among patients with chronic disease and in the elderly.

One of the most important novel serotypes, Ad14, previously rarely reported, is now considered as an emerging Ad type causing severe and sometimes fatal respiratory illness in patients of all ages (45). Beginning in 2005, Ad14 cases were suddenly identified in four locations across USA (46); the strain associated with this outbreak was different than the original Ad14 strain isolated in 1950s. The novel strain, Ad14a, has now spread to numerous US states and is associated with a higher rate of severe illness when compared to other Ad strains.

Novel Ad species have also been recently detected in cross-species infections from non-human primates to man in USA and between psittacine birds and man in China (47). These cross-species infections indicate that Ads should be monitored for their potential to cause cross-species outbreaks. In a recent review of the risks of potential outbreaks associated with zoonotic Ad (48), it was noted that intense human–animal interaction is likely to increase the probability of emergent cross-species Ad infection. Additionally, the recombination of AdVs with latent “host-specific” AdVs is the most likely scenario for adaptation to a new host, either human or animal.

Currently, there are no FDA approved antivirals for Ad infection; however, the best antiviral success has been seen with ribavirin, cidofovir, and most recently brincidofovir an analog of cidofovir (49).

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.

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.

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.

In this study, which is aimed at identifying anti-CPV drugs for potential therapeutic use, a CPE-based assay was developed for screening CPV inhibitors from an FDA-approved drug library. After screening, the top three drugs, Nitazoxanide, Closantel Sodium, and Closantel, with higher percentage CPE inhibition, were selected and further confirmed by qPCR and IFA. In addition, the identified drugs can inhibit different subspecies of CPV variants and displayed broad-spectrum antiviral activity against CPV. Hence, these drugs may provide potential options for the treatment of CPV infection.

Myxoviruses are enveloped, negative-strand RNA viruses that are transmitted through the respiratory route. The orthomyxovirus family comprises five different genera of which the influenza viruses are clinically most relevant. Of the paramyxoviridae, respiratory syncytial virus (RSV), measles virus (MeV), mumps virus (MuV), human parainfluenzaviruses (HPIV) and the recently emerged, highly pathogenic zoonotic henipaviruses constitute major human pathogens. Although clinical complications associated with some myxoviruses involve persistent infections, the viruses predominantly induce acute respiratory or systemic disease.

Collectively, myxoviruses are responsible for the majority of human morbidity and mortality due to viral respiratory illness globally,. In particular, influenza virus is the leading cause of morbidity and mortality from respiratory disease in North America despite the existence of vaccine prophylaxis. This is due to the fact that the vaccines currently in use reduce illness in approximately 70% of healthy adults when homologous to the prevalent circulating virus, but protection in the elderly reaches only approximately 40%. Vaccine efficacy is reduced substantially when the circulating strains differ from those constituting the vaccine.

Despite extensive research and in contrast to, for instance, MeV and MuV, no vaccines are currently available against several major pathogens of the paramyxovirus family such as RSV or different HPIVs. Infection with RSV is the leading cause of pneumonia and bronchiolitis in infants, both associated with significant mortality, while HPIV types 1 and 2 are the primary cause of croup syndrome and can likewise result in serious lower respiratory diseases such as pneumonia and bronchiolitis,.

The availability of effective antiviral therapy for most clinically significant myxovirus infections is limited. Licensed neuraminidase inhibitors for influenza therapy, Zanamivir and Oseltamivir, show efficacy when administered within a 48-hour window after the onset of symptoms, but are increasingly compromised by pre-existing or emerging viral resistance,,. Ribavirin, although approved for RSV treatment, shows limited utility due to efficacy and toxicity issues. The polyclonal immunoglobulin RSV-IVIG and the humanized monoclonal antibody Synagis provide RSV prophylaxis, but use is limited to high-risk pediatric patients. Considering the high mutation rates seen in particular with RNA viruses,, the development of novel types of myxovirus inhibitors that circumvent the rapid development of resistance is highly desirable.

Of the strategies conceivable towards this goal, targeting host factors required for completion of the viral life cycle rather than pathogen-encoded factors directly has received heightened interest in recent years,. This approach is expected to establish a significant barrier against spontaneous viral escape from inhibition, since individual viral mutations are less likely to compensate for the loss of an essential host cofactor than to prevent high-affinity binding of a conventional, pathogen-directed antiviral. Given some degree of overlap of host cell pathways required for successful replication of related viral pathogens, host-directed antiviral approaches also have the potential to move beyond the one-bug one-drug paradigm by broadening the pathogen target range of a chemical scaffold.

Naturally, targeting host factors for antiviral therapy bears an inherently higher potential for undesirable drug-induced side effects than conventional pathogen-directed strategies. While the approach is nevertheless under investigation for the treatment of chronic viral infections such as HSV-1 and HIV-1,, an application to the inhibition of infections by pathogens predominantly associated with severe acute disease, such as most members of the myxovirus families, is anticipated to render drug-related side effects tolerable to some extent, since the necessary treatment time and concomitant host exposure to the drug remain limited. In the case of influenza infections, for instance, typical neuraminidase inhibitor regimens consist of twice daily administration for a five-day period for treatment, or a 10-day period for prophylaxis.

Relying on a broadened anti-myxovirus target spectrum as the main selection criterion in secondary screening assays, we have mined results of a recently completed high throughput chemical library screen to identify hit candidates with a possible host-directed mechanism of action. This has yielded a compound class with broad anti-viral activity, which was subjected to synthetic scaffold optimization, quantification of active concentrations for a select group of clinically relevant ortho- and paramyxovirus family members, testing against a panel of exposed host cells of different species origin, and characterization of the compound-induced point-of-arrest in viral life cycle progression. Viral adaptation to growth in the presence of inhibitor has been employed to compare escape rates from inhibition by this new compound class with those from a well-characterized, pathogen-directed antiviral.

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.

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.

For detection of antibodies against betacoronaviruses, 30 acute serum samples as well as the corresponding sera (obtained 2 to 3 weeks later) of the convalescent dogs were examined by an indirect immunofluorescence test. Madin‐Darby bovine kidney cells were disseminated on 96‐well microtitre plates (100 μL/well) and then incubated at 37°C in a humid 5% CO2‐atmosphere overnight. After washing the plates with phosphate‐buffered saline (PBS) solution, the adherent cells were infected with BCoV strain 15317/82 and incubated at 37°C for 48 hours. Subsequently cells were washed with PBS again and fixed with 100 mL of 96% ethanol.

The sera underwent twofold serial dilutions from 1:20 to 1:5120 with PBS and immunofluorescence test was performed as follows:

Ethanol was discharged, and the 96‐well microtitre plates were washed three times with PBS; 50 μL of the previously diluted sera per well were added and incubated at 37°C for 30 minutes. Thereafter, the plates were washed three times with PBS and 50 μL of 1:40 diluted fluorescein isothiocyanate (FITC)‐conjugate (anti‐dog IgG, Jackson) was added to each well. After incubation at 37°C for another 30 minutes and three washing cycles with PBS, 50 μL/well Eriochrome black T indicator (diluted 1:200 with PBS) was filled in each well of the 96‐well microtitre plate to reduce background fluorescence. Plates incubated for 5 minutes at room temperature before cells were washed three times with PBS once more. Finally, wells were filled with 50 μL/well of glycerine buffer solution to prevent the cells from drying.

For evaluation of the microtitre plates an inverse ultraviolet microscope was used. The highest dilution with a clear cytoplasmatic fluorescence was equivalent to the specific antibody titre of each serum sample. Samples that showed no fluorescence in dilution 1:20 were regarded as negative (no antibodies present). Each assay included a positive and a negative control serum.

While it is often recommended that a detailed understanding of dog ecology is needed for effective canine rabies control, the consistency of research findings generated over the past 30 years allows us to be confident in concluding that mass dog vaccination is feasible across a wide range of settings and campaigns can and should be initiated without delay. In some cases, more nuanced understanding may be required to improve coverage, but these insights can be often be gained through implementation of control measures and used to progressively improve the design and delivery of subsequent interventions. Key considerations include the nature and degree of community engagement, timing of campaigns, placement of vaccination stations and whether or not to charge owner fees [62–64]. The costs of implementing campaigns free of charge may exceed those readily available to government veterinary services, but many approaches can still be explored to improve affordability, acceptability and cost-effectiveness.

While there is widespread agreement about the central importance of mass dog vaccination in canine rabies control and elimination, the role of dog population management remains the subject of debate. There is a rich literature around fertility control for management of roaming dog and wildlife populations. However, as rabies transmission varies little with dog density, reproductive control measures carried out with the aim of reducing dog density are not likely to be effective for rabies control. In theory, reducing population turnover (e.g. through improving life expectancy and/or reducing fecundity) could help sustain population immunity between campaigns and improve cost-effectiveness. However, there is little empirical evidence that dog population management tools have been able to achieve this. Furthermore, even in populations with a high turnover, achieving a 70% coverage during annual campaigns has been sufficient to sustain population immunity above critical thresholds determined by R0. The relatively high cost of sterilization also means that strategies which combine vaccination and sterilization are less cost-effective in terms of achieving human health outcomes than strategies based on dog vaccination alone, even in populations with a large proportion of roaming dogs. Improved dog population management is undoubtedly a desirable longer-term goal for animal health and welfare and may have important secondary benefits for rabies control, for example by enhancing community or political support. However, a focus on mass dog vaccination currently remains the most pragmatic and cost-effective approach to canine rabies control and elimination.

The limited availability and quality of routine animal rabies surveillance data in LMICs has been an obstacle to the application of the analytical approaches from which we have learned so much about wildlife rabies. ‘Gold standard’ surveillance data based on laboratory-confirmed diagnosis is hampered not only by limited laboratory infrastructure but also by the practical challenges of locating, sampling and submitting specimens. However, pragmatic approaches to improving rabies surveillance have yielded rich insights. In addition to providing a foundation for burden of disease estimates, data on animal-bite injuries have been a used as a reliable indicator of canine rabies incidence, revealing new understanding of rabies metapopulation dynamics, as well as improving detection of animal rabies cases, the management of animal bites and the cost-effectiveness of PEP.

Pragmatic solutions are also being found to improve rabies diagnosis in settings with limited laboratory infrastructure, including techniques to support decentralized laboratory testing (e.g. direct rapid immunohistochemical test, dRIT) [73–76] and field diagnosis (e.g. immunochromatographic tests) [77–79]. These have great potential for empowering field staff to engage in rabies surveillance and respond more effectively to surveillance data, but standardization and quality control of field diagnostic kits still needs improvement. Given the rapid advances in metagenomic sequencing methods, future approaches may include real-time genomic surveillance. However, even simple technologies such as mobile phones can serve as leapfrogging technology that can dramatically improve the extent and resolution of rabies surveillance data.

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.

All RNA extracts were subjected to a previously-established RT-PCR assay for detection of CnPnV RNA, with minor modifications. Briefly, a one-step method was adopted using SuperScript™ One-Step RT-PCR for Long Templates (Invitrogen srl, Milan, Italy), according to the manufacturer’s instructions, and primers SH1F/SH187R that amplify a 208-bp of the small hydrophobic (SH) protein gene (Table 1). The following thermal protocol was used: reverse transcription at 50°C for 30 min, inactivation of Superscript II RT at 94°C for 2 min, 40 cycles of 94°C for 30 s, 54°C for 30 s, 68°C for 60 s, with a final extension at 68°C for 10 min. The PCR products were detected by electrophoresis through a 1.5% agarose gel and visualisation under UV light after ethidium bromide staining.

In addition to the gel-based RT-PCR, a real-time RT-PCR assay based on the TaqMan technology was developed for the rapid detection and quantification of the CnPnV RNA in all clinical samples. Reactions were carried out using Platinum® Quantitative PCR SuperMix-UDG (Invitrogen srl) in a 50-µl mixture containing 25 µl of master mix, 300 nM of primers CnPnV-For and CnPnV-Rev, 200 nM of probe CnPnV-Pb (Table 1) and 10 µl of template RNA. Duplicates of log10 dilutions of standard RNA were analyzed simultaneously in order to obtain a standard curve for absolute quantification. The thermal profile consisted of incubation with UDG at 50°C for 2 min and activation of Platinum Taq DNA polymerase at 95°C for 2 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 48°C for 30 s and extension at 60°C for 30 s.

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).

Tissue sections from the gastrointestinal tracts of six dogs were deparaffinized through baths of LMR-SOL (1-bromopropane, 2-methylpropane-2-ol, and acetonitrile), followed by rehydration with successive baths of 100, 90, 70, and 50% ethanol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in PBS. Nonspecific binding was blocked with 3% bovine serum albumin (BSA) in PBS. H and A antigen detection was then performed as previously reported (23). To assess the ability of VLPs to bind to tissue sections, after blocking, 1 μg/ml VLPs was incubated with the sections overnight at room temperature. Anti-HuNoV primary antibody was then incubated with the tissue sections for 1 h at 37°C. After three washes in PBS, sections were incubated with secondary anti-rabbit biotinylated antibody (Vector Laboratories, Burlingame, CA) diluted in 1% BSA in PBS for 1 h. Sections were washed three times in PBS prior to addition of HRP-conjugated avidin D (Vector Laboratories, Burlingame, CA) also diluted in 1% BSA in PBS. Substrate was added to the slides (AEC kit; Vector Laboratories, Burlingame, CA), followed by Mayer's hematoxylin solution (Merck, Whitehouse Station, NJ) for contrast staining.

The virus consensus sequences were compared with the online NCBI Genbank using BLASTN and the closest related sequences selected for further analyses using MEGA 7 software43. For the canine parvovirus 2 (CPV2) comparisons, we also added the available sequences of the CPV2 Protech C3 Vaccine and CPV2 Canigen C3 Vaccine viruses20. Sequences were aligned using Clustal-W44 or in some cases for codon alignments using Muscle45. The Maximum likelihood (ML) method with the best fitting model, as determined by MEGA, was selected for generating phylogenetic trees and the robustness of different nodes assessed by bootstrap analysis using 1000 replicates for nucleotide alignments and 500 replicates for amino acid alignments.

Recent research on rabies has generated a strong body of evidence for the feasibility of elimination of canine rabies through mass vaccination of domestic dogs. Global momentum is now building towards implementation of large-scale programmes to achieve first, the elimination of human deaths mediated by canine rabies, and second, disruption of transmission within the dog population and the elimination of canine rabies entirely. However, time is short to reach these global targets and there is no cause for further delay.

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.

CPV is a widely distributed virus and contains at least three main subspecies: CPV-2a, CPV-2b, and CPV-2c. Currently, commercial vaccines cannot provide complete protection against all CPV variants. Moreover, no effective drug is available to control CPV infection except for supportive and symptom-based care. Hence, it is important to develop an alternative treatment against CPV infection. In this study, we developed a CPE-based assay to screen CPV inhibitors from a FDA-approved drug library, and successfully identified three FDA-approved CPV inhibitors. These drugs might provide potential treatment options for anti-CPV infections.

Although the selectivity index (SI) of Gemcitabine HCl, Cladribine, Gemcitabine, and Trifluridine were at a higher level (Table 1), the percentage CPE inhibition of the four drugs was always maintained at a lower level (Figure S1). The maximum percentage CPE inhibitions for the drugs were between 51.80 ± 2.48 and 68.37 ± 7.79, which are relatively lower CPE inhibition levels that would not increase with increased drug concentration. Therefore, Nitazoxanide, Closantel Sodium, and Closantel were selected for further study.

As mentioned above, the identified drugs Nitazoxanide, Closantel Sodium, and Closantel can reduce the copy numbers of CPV viral DNA to 0.07%, 24.04%, and 20.83%, respectively, compared with the 0.1% DMSO-treated control (Figure 3). Meanwhile, the IFA result also showed that these identified drugs inhibited CPV infection in a dose-dependent reduction manner. Western blot showed that 10 μM Nitazoxanide treatment reduced relative VP2 expression in three CPV variants SD6, SD3, and BJ-1 to 9.68%–36.29%, and the reduction rates following 10 μM Closantel Sodium and Closantel treatment were 12.83%–23.85% and 10.78%–30.1%, respectively (Figure 6). These results indicated that the identified drugs had significant inhibitory effects against CPV infection in F81 cells.

In previous studies, two drugs Oseltamivir and Cidofovir were added in the second round of screening. As a neuraminidase (NA) inhibitor, Oseltamivir has been used to treat the human influenza virus. Savigny and Macintire (2010) used Oseltamivir for CPV enteritis and found that the Oseltamivir-treated group gained a significant increase of weight and had no changes in white blood cell (WBC) count compared to the control group; however, the authors also reported that no obvious advantage had been established. Cidofovir is a broad-spectrum anti-DNA virus drug, which had been evaluated for the treatment of human papillomavirus (HPV)-associated tumors. Our CPE-based screening assay showed that the percentage CPE inhibition of Oseltamivir and Cidofovir were 2.13 ± 2.41 and −1.28 ± 1.03 (Table S1), respectively, and that these two drugs had no anti-CPV effects on F81 cells.

Previously, Nitazoxanide was used to treat cryptosporidiosis, giardiasis, and other parasitic infections. Recently Nitazoxanide was reported to inhibit various DNA and RNA viruses, including hepatitis B virus (HBV), human cytomegalovirus (HCMV), influenza A virus, hepatitis C virus, norovirus, rotavirus, Japanese encephalitis virus (JEV), coronavirus chikungunya virus (CHIKV), human immunodeficiency virus (HIV), and ZIKV.

The antiviral mechanism of Nitazoxanide remains unclear for now. Nitazoxanide could impair the terminal glycosylation of the influenza A hemagglutinin protein or the formation of E1-E2 (Rubella virus surface glycoproteins) complex of the Rubella virus (RV), thus affecting the assembly of influenza A virus and RV, respectively. In addition, Nitazoxanide could also hinder the interactions between the proteins NSP5 and NSP2 of Rotavirus or the interactions between proteins NS2B and NS3 of ZIKV and dengue virus 2 (DENV2). Mercorelli et al. (2016) also reported that Nitazoxanide can inhibit the transcriptional activation properties of the HCMV immediate-early 2 (IE2) protein. These results indicated the virus-specific effects of Nitazoxanide.

Since Nitazoxanide can inhibit the replication of various DNA and RNA viruses, various studies have focused on identifying host factors to explain the broad antiviral activities of Nitazoxanide. Ashiru et al. (2014) reported that Nitazoxanide depleted intracellular Ca2+ stores, besides the phosphorylation of PKR and eIF2α, further affecting N-linked glycosylation of the bovine viral diarrhea virus (BVDV) E2 protein and trafficking from the ER to the Golgi. Nitazoxanide can elicit antiviral innate immunity and reduce the HIV replication by activating the interferon system and further expression of various interferon-stimulated genes (ISGs). In addition, Nitazoxanide might block the production of acetyl-CoA, which is a required metabolic intermediates for Vaccinia virus (VACV) reproduction. In general, further studies are still required to clearly elucidate the antiviral mechanism of Nitazoxanide.

Closantel sodium and Closantel were also identified and shown to have anti-CPV activities. Closantel is a salicylanilide derivative and is considered to be an anthelmintic agent in livestock. The antiangiogenesis and anticancer effects of Closantel sodium and Closantel have also been previously reported. However, to our knowledge, neither the antiviral activity nor the antiviral mechanisms of Closantel sodium and Closantel have been reported before. Previous studies have shown that Closantel inhibited B-Raf (a serine/threonine kinase) V600E, adenine nucleotide translocase (ANT), SPAK and OSR1 kinase. In addition, Senkowski et al. (2015) reported that Closantel could inhibit mitochondrial respiration as well. These reports may contribute to further studies on the antiviral mechanism of Closantel.

VLPs were heated to 95°C for 5 min in the presence of SDS loading buffer and electrophoresed on 12.5% SDS-polyacrylamide gels. For Coomassie blue staining, the gels were incubated with Coomassie blue for 1 h at room temperature prior to destaining. Proteins were transferred from SDS-polyacrylamide gels to polyvinylidene difluoride membranes for Western blotting. The membranes were blocked for 1 h at room temperature with 5% milk in PBS-T and then incubated overnight at 4°C with canine serum samples diluted 1:1,000. The excess antibody was washed three times in PBS-T and incubated for 1 h with anti-canine IgG secondary antibody conjugated to horseradish peroxidase (Sigma-Aldrich) diluted 1:10,000 in 5% milk–PBS-T. After washing away excess secondary antibody, the bands were detected using enhanced chemiluminescence reagent (GE Healthcare).