Interspecies Transmission Of Viruses Research Articles

Infection Studies with Airway Organoids from Carollia perspicillata Indicate That the Respiratory Epithelium Is Not a Barrier for Interspecies Transmission of Influenza Viruses.

Bats are a natural reservoir for many viruses and are considered to play an important role in the interspecies transmission of viruses. To analyze the susceptibility of bat airway cells to infection by viruses of other mammalian species, we developed an airway organoid culture model derived from airways of Carollia perspicillata. Application of specific antibodies for fluorescent staining indicated that the cell composition of organoids resembled those of bat trachea and lungs as determined by immunohistochemistry. Infection studies indicated that Carollia perspicillata bat airway organoids (AOs) from the trachea or the lung are highly susceptible to infection by two different porcine influenza A viruses. The bat AOs were also used to develop an air-liquid interface (ALI) culture system of filter-grown epithelial cells. Infection of these cells showed the same characteristics, including lower virulence and enhanced replication and release of the H1N1/2006 virus compared to infection with H3N2/2007. These observations agreed with the results obtained by infection of porcine ALI cultures with these two virus strains. Interestingly, lectin staining indicated that bat airway cells only contain a small amount of alpha 2,6-linked sialic acid, the preferred receptor determinant for mammalian influenza A viruses. In contrast, large amounts of alpha 2,3-linked sialic acid, the preferred receptor determinant for avian influenza viruses, are present in bat airway epithelial cells. Therefore, bat airway cells may be susceptible not only to mammalian but also to avian influenza viruses. Our culture models, which can be extended to other parts of the airways and to other species, provide a promising tool to analyze virus infectivity and the transmission of viruses both from bats to other species and from other species to bats. IMPORTANCE We developed an organoid culture system derived from the airways of the bat species Carollia perspicillata. Using this cell system, we showed that the airway epithelium of these bats is highly susceptible to infection by influenza viruses of other mammalian species and thus is not a barrier for interspecies transmission. These organoids provide an almost unlimited supply of airway epithelial cells that can be used to generate well-differentiated epithelial cells and perform infection studies. The establishment of the organoid model required only three animals, and can be extended to other epithelia (nose, intestine) as well as to other species (bat and other animal species). Therefore, organoids promise to be a valuable tool for future zoonosis research on the interspecies transmission of viruses (e.g., bat → intermediate host → human).

Viral cross-class transmission results in disease of a phytopathogenic fungus

Interspecies transmission of viruses is a well-known phenomenon in animals and plants whether via contacts or vectors. In fungi, interspecies transmission between distantly related fungi is often suspected but rarely experimentally documented and may have practical implications. A newly described double-strand RNA (dsRNA) virus found asymptomatic in the phytopathogenic fungus Leptosphaeria biglobosa of cruciferous crops was successfully transmitted to an evolutionarily distant, broad-host range pathogen Botrytis cinerea. Leptosphaeria biglobosa botybirnavirus 1 (LbBV1) was characterized in L. biglobosa strain GZJS-19. Its infection in L. biglobosa was asymptomatic, as no significant differences in radial mycelial growth and pathogenicity were observed between LbBV1-infected and LbBV1-free strains. However, cross-species transmission of LbBV1 from L. biglobosa to infection in B. cinerea resulted in the hypovirulence of the recipient B. cinerea strain t-459-V. The cross-species transmission was succeeded only by inoculation of mixed spores of L. biglobosa and B. cinerea on PDA or on stems of oilseed rape with the efficiency of 4.6% and 18.8%, respectively. To investigate viral cross-species transmission between L. biglobosa and B. cinerea in nature, RNA sequencing was carried out on L. biglobosa and B. cinerea isolates obtained from Brassica samples co-infected by these two pathogens and showed that at least two mycoviruses were detected in both fungal groups. These results indicate that cross-species transmission of mycoviruses may occur frequently in nature and result in the phenotypical changes of newly invaded phytopathogenic fungi. This study also provides new insights for using asymptomatic mycoviruses as biocontrol agent.

Fatal Human Alphaherpesvirus 1 Infection in Free-Ranging Black-Tufted Marmosets in Anthropized Environments, Brazil, 2012-2019.

Human alphaherpesvirus 1 (HuAHV1) causes fatal neurologic infections in captive New World primates. To determine risks for interspecies transmission, we examined data for 13 free-ranging, black-tufted marmosets (Callithrix penicillata) that died of HuAHV1 infection and had been in close contact with humans in anthropized areas in Brazil during 2012–2019. We evaluated pathologic changes in the marmosets, localized virus and antigen, and assessed epidemiologic features. The main clinical findings were neurologic signs, necrotizing meningoencephalitis, and ulcerative glossitis; 1 animal had necrotizing hepatitis. Transmission electron microscopy revealed intranuclear herpetic inclusions, and immunostaining revealed HuAHV1 and herpesvirus particles in neurons, glial cells, tongue mucosal epithelium, and hepatocytes. PCR confirmed HuAHV1 infection. These findings illustrate how disruption of the One Health equilibrium in anthropized environments poses risks for interspecies virus transmission with potential spillover not only from animals to humans but also from humans to free-ranging nonhuman primates or other animals.

Full Viral Genome Sequencing and Phylogenomic Analysis of Feline Herpesvirus Type 1 (FHV-1) in Cheetahs (Acinonyx jubatus).

Feline herpesvirus type 1 (FHV-1) is endemic in captive cheetahs and sporadically causes devastating disease. Modified live vaccines (MLV), intended for use in domestic cats, are used in some captive cheetah populations and have been anecdotally linked to disease in certain subpopulations. Ten FHV-1 isolates from ten captive cheetahs and one isolate from an MLV used to inoculate four of the host animals were analyzed. Viral DNA was extracted for full-genome sequencing by Illumina MiSeq with viral genomes then used for phylogenomic and recombinational analyses. The FHV-1 shed by vaccinated cheetahs were almost identical to the MLV, with few variants among viral genomes. Eight cheetah FHV-1 isolates and the MLV were grouped in a clade along with FHV-1 isolates from domestic cats in the USA. The remaining two cheetah FHV-1 isolates (unknown host vaccine status) were not associated with a clade. The likely ancestral origin of these two isolates involves recombination events between Australian domestic cat and cheetah FHV-1 isolates. Collectively, these data suggest that the MLV is capable of causing clinical disease and viral shedding in some cheetahs and represents evidence of interspecies transmission of virus between domestic and wild cats.

Genetic Strain Diversity of Multi-Host RNA Viruses that Infect a Wide Range of Pollinators and Associates is Shaped by Geographic Origins.

Emerging viruses have caused concerns about pollinator population declines, as multi-host RNA viruses may pose a health threat to pollinators and associated arthropods. In order to understand the ecology and impact these viruses have, we studied their host range and determined to what extent host and spatial variation affect strain diversity. Firstly, we used RT-PCR to screen pollinators and associates, including honey bees (Apis mellifera) and invasive Argentine ants (Linepithema humile), for virus presence and replication. We tested for the black queen cell virus (BQCV), deformed wing virus (DWV), and Kashmir bee virus (KBV) that were initially detected in bees, and the two recently discovered Linepithema humile bunya-like virus 1 (LhuBLV1) and Moku virus (MKV). DWV, KBV, and MKV were detected and replicated in a wide range of hosts and commonly co-infected hymenopterans. Secondly, we placed KBV and DWV in a global phylogeny with sequences from various countries and hosts to determine the association of geographic origin and host with shared ancestry. Both phylogenies showed strong geographic rather than host-specific clustering, suggesting frequent inter-species virus transmission. Transmission routes between hosts are largely unknown. Nonetheless, avoiding the introduction of non-native species and diseased pollinators appears important to limit spill overs and disease emergence.

The detection of honey bee (Apis mellifera)-associated viruses in ants

Interspecies virus transmission involving economically important pollinators, including honey bees (Apis mellifera), has recently sparked research interests regarding pollinator health. Given that ants are common pests within apiaries in the southern U.S., the goals of this study were to (1) survey ants found within or near managed honey bee colonies, (2) document what interactions are occurring between ant pests and managed honey bees, and 3) determine if any of six commonly occurring honey bee-associated viruses were present in ants collected from within or far from apiaries. Ants belonging to 14 genera were observed interacting with managed honey bee colonies in multiple ways, most commonly by robbing sugar resources from within hives. We detected at least one virus in 89% of the ant samples collected from apiary sites (n = 57) and in 15% of ant samples collected at non-apiary sites (n = 20). We found that none of these ant samples tested positive for the replication of Deformed wing virus, Black queen cell virus, or Israeli acute paralysis virus, however. Future studies looking at possible virus transmission between ants and bees could determine whether ants can be considered mechanical vectors of honey bee-associated viruses, making them a potential threat to pollinator health.

Absence of adaptive evolution is the main barrier against influenza emergence in horses in Asia despite frequent virus interspecies transmission from wild birds.

Virus ecology and evolution play a central role in disease emergence. However, their relative roles will vary depending on the viruses and ecosystems involved. We combined field studies, phylogenetics and experimental infections to document with unprecedented detail the stages that precede initial outbreaks during viral emergence in nature. Using serological surveys we showed that in the absence of large-scale outbreaks, horses in Mongolia are routinely exposed to and infected by avian influenza viruses (AIVs) circulating among wild birds. Some of those AIVs are genetically related to an avian-origin virus that caused an epizootic in horses in 1989. Experimental infections showed that most AIVs replicate in the equine respiratory tract without causing lesions, explaining the absence of outbreaks of disease. Our results show that AIVs infect horses but do not spread, or they infect and spread but do not cause disease. Thus, the failure of AIVs to evolve greater transmissibility and to cause disease in horses is in this case the main barrier preventing disease emergence.

Infection of covert mortality nodavirus in Japanese flounder reveals host jump of the emerging alphanodavirus.

Interspecies transmission of viruses, where a pathogen crosses species barriers and jumps from its original host into a novel species, has been receiving increasing attention. Viral covert mortality disease, caused by covert mortality nodavirus (CMNV), is an emerging disease that has recently had a substantial impact on shrimp aquaculture in Southeast Asia and Latin America. While investigating the host range of CMNV, we found that this virus is also capable of infecting populations of the farmed Japanese flounder Paralichthys olivaceus, a vertebrate host. The infected fish were being raised in aquaculture facilities that were also producing marine shrimp. Through RT-nPCR, targeting the RNA-dependent RNA polymerase (RdRp) gene of CMNV, we found that 29 % of the fish sampled were positive. The amplicons were sequenced and aligned to the RdRp gene of shrimp CMNV and were found to have 98 % identity. Histopathological examination indicated that CMNV-positive fish showed vacuolation of nervous tissue in the eye and brain, as well as extensive necrosis of cardiac muscle. In situ hybridization showed positive reactions in tissues of the eye, brain, heart, liver, spleen and kidney of infected fish. Transmission electron microscopy showed the presence of CMNV-like particles in all of the above-mentioned tissues, except for brain. The novel finding of a shrimp alphanodavirus that can also infect farmed P. olivaceus indicates that this virus is capable of naturally crossing the species barrier and infecting another vertebrate. This finding will contribute to the development of efficient strategies for disease management in aquaculture.

Interspecies transmission of Rotaviruses among ruminants, dogs and humans: Current facts and remarks

Rotaviruses are considered to be a major cause of diarrhoea to humans as well as a wide variety of animals and may cause serious economic losses in livestock animals, especially swine and ruminants. This fact, along with the genetic diversity that characterizes members of the Rotavirus group, raised concerns regarding the potential of virus interspecies transmission among various species of animals and humans. Regarding the presence and the epidemiology of Rotaviruses in ruminants in association with closely related humans and dogs, research is limited and few data have been presented in recent years. In this review we present all the latest information regarding the distribution of genotypes of Rotavirus strains in ruminants, dogs and humans.

Molecular and experimental evidence of Watermelon (Citrullus lanatus) as host of the Mexican variant of Papaya meleira virus

Papaya sticky disease, caused by Papaya meleira virus (PMeV), results in complete crop loss in papaya orchards, because infected fruits are unacceptable for consumption. Little is known about sticky disease epidemiology, and the best available strategy for disease management is to rogue infected plants. Identification of alternative hosts for this virus is important for developing control strategies. In Mexico, papayas and watermelons are often cultivated in close proximity or overlapping cycles, which may favor virus interspecies transmission by putative insect vectors of PMeV. Watermelon seedlings (Citrullus lanatus Thunb.) were inoculated with latex from papayas infected with a Mexican variant of PMeV (PMeV-Mx), and infection was confirmed by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). Amplicons (491 bp) of the RNA-dependent RNA polymerase (RdRp) gene of PMeV-Mx were detected in axillary leaves of all inoculated seedlings seven days post-inoculation (dpi). Circa 10 to 12 dpi, necrotic lesions were observed on the edges of the leaves. Absolute quantification of PMeV-Mx-RNA by RT-qPCR showed an increase in viral load over time (from 0.51 to 586.15 pg/μL), three to 14 dpi. Molecular and experimental evidence demonstrate is also a host for PMeV-Mx and may be an alternative host.

Dispersal and Transmission of Avian Paramyxovirus Serotype 4 among Wild Birds and Domestic Poultry.

Avian paramyxovirus serotype 4 (APMV-4) is found sporadically in wild birds worldwide, and it is an economically important poultry pathogen. Despite the existence of several published strains, very little is known about the distribution, host species, and transmission of APMV-4 strains. To better understand the relationships among these factors, we conducted an APMV-4 surveillance of wild birds and domestic poultry in six provinces of China suspected of being intercontinental flyways and sites of interspecies transmission. APMV-4 surveillance was conducted in 9,160 wild birds representing seven species, and 1,461 domestic poultry in live bird markets (LMBs) from December 2013 to June 2016. The rate of APMV-4 isolation was 0.10% (11/10,621), and viruses were isolated from swan geese, bean geese, cormorants, mallards, and chickens. Sequencing and phylogenetic analyses of the 11 isolated viruses indicated that all the isolates belonging to genotype I were epidemiologically connected with wild bird-origin viruses from the Ukraine and Italy. Moreover, chicken-origin APMV-4 strains isolated from the LBMs were highly similar to wild bird-origin viruses from nearby lakes with free-living wild birds. In additional, a hemagglutination-negative APMV-4 virus was identified. These findings, together with recent APMV-4 studies, suggest potential virus interspecies transmission between wild birds and domestic poultry, and reveal possible epidemiological intercontinental connections between APMV-4 transmission by wild birds.

Influenza study of backyard animals in Georgia

ObjectiveThe purpose of this study was to identify zoonotic influenzaviruses in swine and poultry populations in Georgia and to definetheir pandemic potential.IntroductionAquatic birds are the main reservoirs of influenza viruses,however pigs represent an essential host in virus ecology as they aresusceptible to both avian and human influenza viruses. Circulatingzoonotic influenza (A/H7N9, A/H5N1, and A/H3N2v) viruses couldmutate into forms easily transmissible from human-to-human andbecome a public health concern. Georgia is located along routes usedby migrating birds where different species of aquatic birds are found.In 2006, highly pathogenic influenza virus A/H5N1 was detected intwo wild swans in Adjara (western Georgia). Moreover, in the frameof wild bird surveillance, various subtypes of influenza A viruseswere detected in mallard and gulls in Georgia (Lewis, 2013). Thusdomestic animals in Georgia have a potential chance to contractinfluenza viruses from wild birds.MethodsThe Kakheti region, the leading region in cattle breeding andpoultry production in Georgia, was selected for study. Villages wereselected for door-to-door visits to search for ill backyard animalsshowing influenza-like symptoms. In case of identification of a sickanimal, samples were obtained for laboratory investigations; samplecollection forms were filled out to generate epidemiological data.Cloacal and tracheal swabs were taken from poultry; and pharyngealand nasal swabs were collected from pigs. Each specimen wasscreened for influenza A matrix gene by real-time RT-PCR using aprotocol from the Centers for Disease Control Prevention.ResultsEighty four villages in the Kakheti region were surveyed fordomestic animals with influenza-like illness symptoms. In total,164 specimens were collected from 112 backyard animals in55 households (107 samples were from 55 poultry and 57 sampleswere from 57 pigs). All samples tested negative for Influenza A virusby real time RT-PCR. The questionnaire data revealed that the agerange of both pigs and poultry varied from one month to two years;median and mode were both 1 year. Chickens and ducks primarilyfreely ranged in backyards (67%), while half the number of pigs werekept in closed premises. Equally, 61% of pigs and poultry had contactwith other pigs or poultry within the premises.ConclusionsIn spite of the negative findings, we cannot exclude the circulationof influenza viruses in domestic animals in Georgia. Especially,considering the fact that a domestic duck with influenza A/H10virus was identified during veterinarian training in 2010 in Grigoleti(Black sea cost of Georgia) manifesting no clinical symptoms.Therefore, larger scale studies, including swabbing more backyardanimals without any clinical symptoms are necessary to identify inter-species virus transmission in the country.

Occurrence of similar mycoviruses in pathogenic, saprotrophic and mycorrhizal fungi inhabiting the same forest stand.

Fungal viruses (mycoviruses) are considered to be highly host specific, but phylogenetic analysis supports the occasional occurrence of horizontal transmission between species. We used an extensive sampling strategy to investigate whether similar viruses occur in more than one fungal species of the same forest habitat. Mycelial samples were collected from in-growth mesh bags (N = 259), fruiting bodies (N = 173) and cultured isolates (N = 68) at a forest site where the spatial distribution of viral infections in clonal individuals of the wood decay fungus Heterobasidion parviporum was mapped in detail earlier. The investigation revealed previously known Heterobasidion viruses in ∼2% of the single or pooled mycorrhizal samples from mesh bags, ∼3% of the fruiting body samples and none of the fungal cultures analyzed. Novel virus strains distinct from known Heterobasidion viruses were detected in cultures of ectomycorrhizal fungi (Lactarius tabidus, L. rufus) and saprotrophic fungi (Megacollybia platyphylla, Mucoraceae spp.). Overall, our results support the view that mycoviruses do not readily cross species borders. Regarding potential virocontrol applications, the introduction of Heterobasidion viruses into natural habitats is not expected to cause a major infection pressure towards the indigenous fungal community. However, the ecological consequences of the putative interspecies virus transmission events detected require further investigation.

Clinico-molecular diagnosis and phylogenetic investigation of foot-and-mouth disease in small ruminant population of India

Small ruminants (sheep and goats) are susceptible to foot-and-mouth disease (FMD), while studies with due emphasis on their role in the disease epidemiology have been meagre. The present study summarizes the results of clinico-molecular investigation of FMD in sheep and goats across several states of India during 2008–2014, where a total of 51 clinical epithelial tissue samples (vesicle/tongue/gum/foot epithelium) from sheep and 78 from goats were found positive for FMD virus (FMDV) serotype O in serotyping ELISA and multiplex reverse transcription-PCR. The VP1 region-based phylogenetic analysis demonstrated the involvement of O/ME-SA/Ind2001 lineage of serotype O virus in the outbreaks. The field viruses recovered from both small and large ruminant population during the same time period showed a close genetic relationship suggesting frequent inter-species transmission of virus. Since the disease often remains clinically camouflaged in small ruminants, the animals silently infected with FMD may pose potential threats to the in-contact livestock. Regular vaccination combined with surveillance and monitoring of protective antibody status in these species is therefore crucial to the effective control of FMD in the country.

One health, multiple challenges: The inter-species transmission of influenza A virus

Influenza A viruses are amongst the most challenging viruses that threaten both human and animal health. Influenza A viruses are unique in many ways. Firstly, they are unique in the diversity of host species that they infect. This includes waterfowl (the original reservoir), terrestrial and aquatic poultry, swine, humans, horses, dog, cats, whales, seals and several other mammalian species. Secondly, they are unique in their capacity to evolve and adapt, following crossing the species barrier, in order to replicate and spread to other individuals within the new species. Finally, they are unique in the frequency of inter-species transmission events that occur. Indeed, the consequences of novel influenza virus strain in an immunologically naïve population can be devastating. The problems that influenza A viruses present for human and animal health are numerous. For example, influenza A viruses in humans represent a major economic and disease burden, whilst the poultry industry has suffered colossal damage due to repeated outbreaks of highly pathogenic avian influenza viruses. This review aims to provide a comprehensive overview of influenza A viruses by shedding light on interspecies virus transmission and summarising the current knowledge regarding how influenza viruses can adapt to a new host.

Feline immunodeficiency virus and feline leukemia virus infection in free-ranging guignas (Leopardus guigna) and sympatric domestic cats in human perturbed landscapes on Chiloé Island, Chile.

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are two of the most common viruses affecting domestic cats (Felis catus). During the last two decades, reports show that both viruses also infect or affect other species of the family Felidae. Human landscape perturbation is one of the main causes of emerging diseases in wild animals, facilitating contact and transmission of pathogens between domestic and wild animals. We investigated FIV and FeLV infection in free-ranging guignas (Leopardus guigna) and sympatric domestic cats in human perturbed landscapes on Chiloé Island, Chile. Samples from 78 domestic cats and 15 guignas were collected from 2008 to 2010 and analyzed by PCR amplification and sequencing. Two guignas and two domestic cats were positive for FIV; three guignas and 26 domestic cats were positive for FeLV. The high percentage of nucleotide identity of FIV and FeLV sequences from both species suggests possible interspecies transmission of viruses, facilitated by increased contact probability through human invasion into natural habitats, fragmentation of guigna habitat, and poultry attacks by guignas. This study enhances our knowledge on the transmission of pathogens from domestic to wild animals in the global scenario of human landscape perturbation and emerging diseases.

Specificity of pestivirus antibodies in wild ruminants from Switzerland.

Switzerland started a bovine viral diarrhoea (BVD) national eradication campaign in 2008. BVD virus (BVDV) belongs together with classical swine fever virus (CSFV) of pigs and border disease virus (BDV) of sheep to the genus Pestivirus. Ruminant pestiviruses are not strictly hostspecifi c and interspecies virus transmission is possible (Vil ek and Nettleton, 2006). In Switzerland, the shared use of Alpine pastures by multiple cattle herds during the summer grazing season is a widespread tradition. In this practice, cattle can share the grazing grounds with wild ruminants; these may be recipients as well as donors of pestiviruses. In the Pyrenees, for example, massive losses in chamois due to infection with border disease virus are reported (Arnal et al., 2004). In the French and Italian Alps, pestivirus antibodies have been detected in chamois, ibex, roe and red deer, moufl ons and wild boar (Olde Riekerink et al., 2005; Fernandez et al., 2011). In order to determine if wild ruminants may play a role in pestivirus epidemiology in Switzerland, a virological and serological survey was carried out in 2009 – 2011 (Casaubon et al., 2012). Results revealed very low seroprevalences in Alpine chamois (Rupicapra rupicapra rupicapra), Alpine ibex (Capra ibex ibex) and red deer (Cervus elaphus elaphus) of 2.1 %, 1.8 % and 2.7 %, respectively. The determination of the specifi city of pestivirus antibodies, i. e. against which pestivirus species they are directed, can give indication on the origin of the infection and therefore enhance our knowledge on interspecies transmissions of pestiviruses to wild ruminants. Therefore, the present study was raised with the aim to specify the antibodies detected in red deer, Alpine chamois and ibex by using a comparative serum neutralization test (SNT).

Understanding restriction factors and intrinsic immunity: insights and lessons from the primate lentiviruses.

Primate lentiviruses include the HIVs, HIV-1 and HIV-2; the SIVs, which are endemic to more than 40 species of nonhuman primates in Africa; and SIVmac, an AIDS-causing pathogen that emerged in US macaque colonies in the 1970s. Because of the worldwide spread of HIV and AIDS, primate lentiviruses have been intensively investigated for more than 30 years. Research on these viruses has played a leading role in the discovery and characterization of intrinsic immunity, and in particular the identification of several antiviral effectors (also known as restriction factors) including APOBEC3G, TRIM5α, BST-2/tetherin and SAMHD1. Comparative studies of the primate lentiviruses and their hosts have proven critical for understanding both the evolutionary significance and biological relevance of intrinsic immunity, and the role intrinsic immunity plays in governing viral host range and interspecies transmission of viruses in nature.

Substitutions near the Hemagglutinin Receptor-Binding Site Determine the Antigenic Evolution of Influenza A H3N2 Viruses in U.S. Swine

Swine influenza A virus is an endemic and economically important pathogen in pigs, with the potential to infect other host species. The hemagglutinin (HA) protein is the primary target of protective immune responses and the major component in swine influenza A vaccines. However, as a result of antigenic drift, vaccine strains must be regularly updated to reflect currently circulating strains. Characterizing the cross-reactivity between strains in pigs and seasonal influenza virus strains in humans is also important in assessing the relative risk of interspecies transmission of viruses from one host population to the other. Hemagglutination inhibition (HI) assay data for swine and human H3N2 viruses were used with antigenic cartography to quantify the antigenic differences among H3N2 viruses isolated from pigs in the United States from 1998 to 2013 and the relative cross-reactivity between these viruses and current human seasonal influenza A virus strains. Two primary antigenic clusters were found circulating in the pig population, but with enough diversity within and between the clusters to suggest updates in vaccine strains are needed. We identified single amino acid substitutions that are likely responsible for antigenic differences between the two primary antigenic clusters and between each antigenic cluster and outliers. The antigenic distance between current seasonal influenza virus H3 strains in humans and those endemic in swine suggests that population immunity may not prevent the introduction of human viruses into pigs, and possibly vice versa, reinforcing the need to monitor and prepare for potential incursions. Influenza A virus (IAV) is an important pathogen in pigs and humans. The hemagglutinin (HA) protein is the primary target of protective immune responses and the major target of vaccines. However, vaccine strains must be updated to reflect current strains. Characterizing the differences between seasonal IAV in humans and swine IAV is important in assessing the relative risk of interspecies transmission of viruses. We found two primary antigenic clusters of H3N2 in the U.S. pig population, with enough diversity to suggest updates in swine vaccine strains are needed. We identified changes in the HA protein that are likely responsible for these differences and that may be useful in predicting when vaccines need to be updated. The difference between human H3N2 viruses and those in swine is enough that population immunity is unlikely to prevent new introductions of human IAV into pigs or vice versa, reinforcing the need to monitor and prepare for potential introductions.

Bicolored White-toothed Shrews as Reservoir for Borna Disease Virus, Bavaria, Germany

To the Editor: Borna disease (BD) is a fatal neurologic disorder in horses and sheep. The etiologic agent, Borna disease virus (BDV), belongs to the order Mononegavirales, which is composed of many reservoir-bound, highly pathogenic, and zoonotic viruses. To investigate whether small mammals, especially bicolored white-toothed shrews (Crocidura leucodon), which act as BDV reservoirs in Switzerland (1,2), harbor BDV in disease-endemic areas in Bavaria, Germany, we screened 120 small mammals (53 from the family Cricetidae, 41 from the family Muridae, and 26 from the family Soricidae) (Table). We also determined whether BDV infections in small mammals might have different disease courses and whether shrew-to-horse virus transmission occurs. Table Small mammals from 7 stables tested for Borna disease virus infection, Bavaria, Germany, 1997–2012* The small mammals were captured during pest control efforts in stables in Upper Bavaria and Swabia that had a history of acute equine BD during 1997–2012. These stables also had a high probability for presence of C. leucodon shrews as documented by a recent distribution model (3). BDV-specific serum antibodies were identified by using an indirect immunofluorescence test and blood samples or thoracic or abdominal effusions as described (4). Antibodies against BDV were found in 8/105 specimens (Table) at serum dilutions ranging from 1:40 for Mus musculus mouse #1008, 1:80 for M. musculus mouse #1014, 1:2,560 for C. leucodon shrew #5063, 1:10,240 for C. leucodon shrew #2001, and 1:20,480 for C. leucodon shrew #5017. Amplification of viral RNA was conducted by using real-time reverse transcription PCR (RT-PCR) (5) or nested RT-PCR (6) on 119/120 brain samples. In 2/4 BDV-seropositive C. leucodon shrews (#2001 and #5017) BDV RNA was amplified from the brain. The remaining 117 mice and insectivores were negative for BDV RNA, including 6/8 BDV-seropositive animals (Table). Histologic and immunohistochemical (IHC) analyses for detection of BDV antigen were performed for small mammals that had antibodies against BDV or BDV RNA. In addition, histologic and IHC analyses were used to test 36/112 small mammals negative for BDV (by indirect immunofluorescence test and RT-PCR), including 15/16 C. leucodon shrews from 3 stables (B, C, and E), in which BDV-positive mammals were captured. None of the small mammals showed obvious gross or histologic lesions, even in the brain. IHC analysis was performed by using monoclonal antibody Bo18 against BDV nucleoprotein (BDV-N) as described (7). The 2/2 C. leucodon shrews (#2001 and #5017) harboring viral RNA had BDV antigen in the central and peripheral nervous system (brain, spinal cord, spinal trigeminal ganglia, and peripheral nerves). Immunostaining of the skin showed evidence of BDV infection, mainly in epidermal keratinocytes and sebaceous glands, as well as in squamous epithelium and connective tissue of the esophagus. In shrew #5017, renal tubuli and glomeruli, as well as nuclei of bronchiolar epithelial cells, had BDV-N. No evidence for viral antigen was found in the other 42/44 small mammals tested. In situ hybridization was performed by using established protocols (8). Viral genomic RNA and mRNA encoding for the BDV-N gene were found in the brain, spinal cord, ganglia, parotid gland, and sebaceous glands of the skin of 2 shrews positive for BDV by RT-PCR. Thus, BDV antigen and RNA were found in nervous tissue and peripheral organs of 2 C. leucodon shrews, as reported for shrews in Switzerland (1,2). Viral dissemination into peripheral organs represents a prerequisite for successful viral excretion and transmission to other susceptible species. Simultaneous detection of viral genomic RNA and mRNA can indicate viral replication and transcription in peripheral organs. RNA from brains of the 2 BDV-positive C. leucodon shrews (#2001 and #5017) and from 1 horse that had BD and lived in the same stable as shrew #5017 was sequenced as described (2). Comparison of BDV sequence (GenBank accession no. {type:entrez-nucleotide,attrs:{text:KF275185,term_id:548786820,term_text:KF275185}}KF275185) from C. leucodon shrew #5017 with sequence (GenBank accession no. {type:entrez-nucleotide,attrs:{text:KF275184,term_id:548786799,term_text:KF275184}}KF275184) from the affected horse showed 100% identity in a 2,150-nt region (nt 17–2161 covering the N, X, and P genes and half of the M gene). Moreover, the BDV sequence showed 98% homology with those of the BDV isolates of the Baden-Wurttemberg and Bavaria II group (9). The 2 BDV-positive shrews were trapped in April (#2001) and July (#5017) 2012 in different stables in the feeding area for hay (B for #2001) or in the storage area for feed (stable E for #5017), which probably indicates that this food was contaminated with BDV. Viral shedding in shrews might occur from skin, kidney, or gastrointestinal tract, which is similar to shedding by persistently infected, immunotolerant, neonatal Lewis rats (10). In conclusion, BDV RNA, viral antigen, and serum antibodies against BDV were detected in 2/20 C. leucodon shrews, indicating that this shrew is reservoir of BDV in Bavaria. Whether seropositivity without other evidence of BDV infection indicates different courses of infection in small mammals, as known for horses, is not known and warrants further investigation. The absolute homology of shrew and equine BDV suggests successful interspecies virus transmission. Our study provides reliable evidence that C. leucodon shrews acts as reservoirs for BDV in disease-endemic areas in Bavaria, Germany, argues for a general role of this shrew as a reservoir for mammalian bornaviruses.