H5 Avian Influenza Viruses Research Articles

Development of a Hemagglutination Inhibition Assay for Duck Tembusu Virus.

The HB strain of duck Tembusu virus (DTMUV) propagated in the brains of newborn mice was used to prepare antigens for use in the hemagglutination inhibition (HI) test. Results showed that such prepared antigens are highly specific to the serum samples derived from DTMUV-infected animals. No spurious hemagglutination reactions against serum samples specific to avian influenza virus H5, H7, H9 subtypes, Newcastle disease virus, egg drop syndrome virus, duck plague virus, and duck hepatitis A virus were observed. The HI test can detect specific antibodies in the serum samples as early as day 4 after experimental infection of ducks with DTMUV. When compared to a virus neutralization test, the sensitivity is 100%. Overall, the HI test developed is highly specific to DTMUV and can be used in clinical diagnosis of diseases and in vaccine studies to monitor the kinetics of antibody response.

Duck viral infection escalated the incidence of avian pathogenic Escherichia coli in China.

Avian pathogenic Escherichia coli (APEC) causes high mortality in poultry flocks and often is complicated with viral infections, leading to large economic losses; however, little information is available on the epidemiological characteristics of this pathogen in ducks. Therefore, a systemic epidemiological investigation was performed on 325 duck farms from 13 provinces in China during the period of 1 April 2016 until 31 March 2018, covering 2years. A total of 26 APEC strains were isolated from different farms in this study, and analysis showed that all of those isolates carried multiple virulence-associated genes and drug-resistance genes, which led to high pathogenicity (15/26), strong or moderate biofilm formation (24/26) and multidrug-resistant abilities (26/26). On the other hand, coinfection with APEC, H9 avian influenza virus (AIV) and Tembusu virus (TMUV) was very common on those farms (11/26), with APEC and TMUV sharing a similar morbidity peak (from May to September) and susceptibility (60% infections occurred in ducklings); thus, we speculated that the emerging TMUV infection escalated the APEC incidence in ducks. Finally, the data presented in this report enhance the current understanding of the epidemiology of APEC and different viral infections in ducks and provide additional insight into the critical factors that determine their pathogenicity. Meanwhile, the emergence of multidrug-resistant APEC strains and their coinfection with different viruses emphasize that preventive measures against such infections on poultry farms should be implemented immediately.

Molecular and serological prevalence of influenza A viruses in poultry and poultry farmers in the Ashanti region, Ghana

ABSTRACTFor an analysis of the prevalence of influenza A viruses (IAVs) circulating in chickens and their farmers in the Ashanti region, Ghana, we examined 2,400 trachea and cloaca swabs (chickens) and 102 oropharyngeal swabs (farmers) by qRT-PCR. Sera from 1,200 (chickens) and 102 (farmers) were analysed for IAV antibodies by ELISA and haemagglutination inhibition (HI). Avian influenza virus (AIV) was detected in 0.2% (n = 5) of chickens but not farmers. Virus detection was more pronounced in the cloacal (n = 4, 0.3%) than in tracheal swabs (n = 1, 0.1%). AIV antibodies were not detected in chickens. Two farmers (2.0%) tested positive to human seasonal IAV H1N1pdm09. Sixteen (15.7%) farmers tested seropositive to IAV of which 68.8% (n = 11) were due to H1N1pdm09-specific antibodies. AIV H5- or H7-specific antibodies were not detected in the farmers. Questionnaire evaluation indicated the rare usage of basic personal protective equipment by farmers when handling poultry. In light of previous outbreaks of zoonotic AIV in poultry in Ghana the open human-animal interface raises concern from a OneHealth perspective and calls for continued targeted surveillance.

Ecuadorian mainland industrial poultry production is free of H5/H7 Avian influenza virus: National surveillance program in 2016.

Avian influenza (AI) is a disease caused by influenza viruses type A that belong to the Orthomyxoviridae family. AI induces high economic losses in poultry production worldwide. Due to a possible outbreak, a national surveillance program was needed. From April to July 2016, 152 industrial poultry farms were randomly sampled. All samples were analyzed by competitive ELISA for Influenza type A viruses. Suspicious and positive sera were further analyzed by Hemagglutination Inhibition (HI) in order to serotype H5 or H7 low pathogenic avian influenza virus (LPAIV). The farms sampled showed 94.08%, 3.95% and 1.97% of negative, positive and suspicious results, respectively. However, serotyping revealed all positive and suspicious samples were negative to H5/H7 LPAIV. Our results show the absence of AI in the mainland Ecuadorian industrial poultry production.

A review of H5Nx avian influenza viruses.

Highly pathogenic avian influenza viruses (HPAIVs), originating from the A/goose/Guangdong/1/1996 H5 subtype, naturally circulate in wild-bird populations, particularly waterfowl, and often spill over to infect domestic poultry. Occasionally, humans are infected with HPAVI H5N1 resulting in high mortality, but no sustained human-to-human transmission. In this review, the replication cycle, pathogenicity, evolution, spread, and transmission of HPAIVs of H5Nx subtypes, along with the host immune responses to Highly Pathogenic Avian Influenza Virus (HPAIV) infection and potential vaccination, are discussed. In addition, the potential mechanisms for Highly Pathogenic Avian Influenza Virus (HPAIV) H5 Reassorted Viruses H5N1, H5N2, H5N6, H5N8 (H5Nx) viruses to transmit, infect, and adapt to the human host are reviewed.

Mutations in the H7 HA and PB1 genes of avian influenza a viruses increase viral pathogenicity and contact transmission in guinea pigs

ABSTRACT Avian influenza A viruses (AIV) of the H7 subtype continue to evolve posing a pandemic threat. However, molecular markers of H7N7 AIV pathogenicity and transmission in mammals remain poorly understood. In this study, we performed a systematic in vitro and in vivo analysis by comparing an H7N7 highly pathogenic AIV and its ferret adapted variant. Passaging an H7N7 AIV in ferrets led to six mutations in genes encoding the viral polymerase complex and the viral surface proteins. Here, we show that mutations in the H7 hemagglutinin gene cause increased pathogenicity in mice. Contact transmission between guinea pigs required additional mutations in the gene encoding the polymerase subunit PB1. Thus, particular vigilance is required with respect to HA and PB1 mutations as predictive molecular markers to assess the pandemic risk posed by emerging H7 avian influenza viruses.

Rapid Detection of Avian Influenza Virus H9 in broilers by using RT-PCR in Al-Qadisiyah Province

Rapid Detection of Avian Influenza Virus H9 in broilers by using RT-PCR in Al-Qadisiyah Province

Two Single Incursions of H7N7 and H5N1 Low Pathogenicity Avian Influenza in U.K. Broiler Breeders During 2015 and 2016.

Low pathogenicity (LP) avian influenza viruses (AIVs) have a natural reservoir in wild birds. These cause few (if any) overt clinical signs, but include H5 and H7 LPAIVs, which are notifiable in poultry. In the European Union, notifiable avian disease (NAD) demands laboratory confirmation with prompt statutory interventions to prevent dissemination of infection to multiple farms. Crucially, for H5 and H7 LPAIVs, movement restrictions and culling limit the further risk of mutation to the corresponding highly pathogenic (HP) H5 and H7 AIVs in gallinaceous poultry. An H7N7 LPAIV outbreak occurred during February 2015 at a broiler breeder chicken premise in England. Full genome sequencing suggested an avian origin closely related to contemporary European H7 LPAIV wild bird strains with no correlates for human adaptation. However, a high similarity of PB2, PB1, and NA genes with H10N7 viruses from European seals during 2014 was observed. An H5N1 LPAIV outbreak during January 2016 affecting broiler breeder chickens in Scotland resulted in rapid within-farm spread. An interesting feature from this case was that although viral tropism occurred in heart and kidney endothelial cells, suggesting HPAIV infection, the H5N1 virus had the molecular cleavage site signature of an LPAIV belonging to an indigenous European H5 lineage. There was no genetic evidence for human adaptation or antiviral drug resistance. The source of the infection was also likely to be via indirect contact with wild birds mediated via fomite spread from the nearby environment. Both LPAIV outbreaks were preceded by local flooding events that attracted wild waterfowl to the premises. Prompt detection of both outbreaks highlighted the value of the "testing to exclude" scheme launched in the United Kingdom for commercial gallinaceous poultry in 2014 as an early warning surveillance mechanism for NAD.

Inactivated H5 Antigens of H5N8 Protect Chickens from Lethal Infections by the Highly Pathogenic H5N8 and H5N6 Avian Influenza Viruses.

IntroductionHighly pathogenic Asian H5-subtype avian influenza viruses have been found in poultry and wild birds worldwide since they were first detected in southern China in 1996. Extensive control efforts have not eradicated them. Vaccination prevents such viruses infecting poultry and reduces the number lost to compulsory slaughter. The study showed the efficacy of inactivated H5 vaccine from the H5N8 virus against highly pathogenic H5N8 and H5N6 avian influenza viruses in chickens.Material and MethodsReverse genetics constructed an H5 vaccine virus using the HA gene of the 2014 H5N8 avian influenza virus and the rest of the genes from A/PR/8/34 (H1N1). The vaccine viruses were grown in fertilised eggs, partially purified through a sucrose gradient, and inactivated with formalin. Chickens were immunised i.m. with 1 μg of oil-adjuvanted inactivated H5 antigens.ResultsSingle dose H5 vaccine recipients were completely protected from lethal infections by homologous H5N8 avian influenza virus and shed no virus from the respiratory or intestinal tracts but were not protected from lethal infections by heterologous H5N6. When chickens were immunised with two doses and challenged with homologous H5N8 or heterologous H5N6, all survived and shed no virus.ConclusionOur results indicate that two-dose immunisations of chickens with H5 antigens with oil adjuvant are needed to provide broad protection against different highly pathogenic H5 avian influenza viruses.

Genetic characteristics of hemagglutinin genes of nine H5 subtype avian influenza viruses in Weining, Guizhou Province during 2015—2017

Objective To investigate the molecular characteristics of H5 subtype avian influenza viruses (AIV) in Weining, Guizhou Province. Methods Nine representative strains were randomly selected from H5 subtype AIV that were identified by real-time PCR in Weining, Guizhou Province from 2015 to 2017. Nucleic acid was extracted from each sample and hemagglutinin (HA) genes were amplified and then sequenced. Homology, genetic evolution and the sites related to pathogenicity, receptor binding regions as well as potential glycosylation of H5 AIV were analyzed by bioinformation software. Results Homology analysis revealed that there was 96.1%-99.9% and 95.7%-100% similarity among the nine strains in nucleotide and amino acid of HA gene, respectively. These strains belonged to two branches, H5-1 and H5-2. The cleavage site motifs were PLREKRRKR↓GLF for five strains in H5-1 branch and PQRERRRKR↓GLF for four strains in H5-2 branch, which made them high pathogenic. QSG and QRG at the key receptor binding sites were found in H5-1 and H5-2 branch strains, respectively. They were responsible for receptor binding specificity of AIV. Mutations of 138Q, 139G and 53K were all detected in the nine strains. 129K, 189T, 140K and 282V mutations were discovered in the five strains of H5-1 branch, while 189N, 140M and 282I mutations were found in the four strains of H5-2 branch. Results of the glycosylation motif analysis showed that six sites were conservative, but there was an addition of 124NHT site in two strains of H5-2 branch isolated in 2017. Conclusion Two high pathogenic H5 subtypes of AIV could be epidemic in Weining, Guizhou Province during 2015 to 2017. Although H5 subtype AIV did not possess specific receptor binding regions like human influenza viruses, they were in continuous variation with an increase in glycosylation motifs, which might enhance their virulence and pathogenicity to human beings. Hence, surveillance and study on the molecular properties of H5 subtype AIV should be strengthened. Key words: H5 subtype avian influenza virus; Hemagglutinin gene; Molecular characteristic

Duck innate immune responses to high and low pathogenicity H5 avian influenza viruses

Ducks are the reservoir host of influenza A viruses, and are permissive for replication of most strains, yet can elicit robust innate immune responses to highly pathogenic strains. Tissue tropism and viral amino acid differences affect virulence, but we have limited knowledge about how viral differences influence the host innate immune response. Here we compare the innate immune response in Pekin ducks to a recombinant highly-pathogenic avian influenza (HPAI) H5N1 virus and a naturally arising attenuated variant of this strain that differs at one amino acid in polymerase A (T515A), as well as ducks infected with two different H5 strains of low pathogenic avian influenza (LPAI). Using qPCR we examined the relative abundance of transcripts for RIG-I and interferon-beta (IFNβ), and downstream interferon stimulated genes (ISGs). The polymerase PA (T515A) mutation did not significantly affect replication in vivo but greatly attenuated host interferon responses. ISG induction was robust for both H5N1 strains, but was three times lower for the PA mutant strain. Low pathogenic viruses elicited detectable induction of RIG-I, IFNβ and ISGs in lung and intestine tissues that correlated with the recovery of viruses from tracheal or cloacal swabs. Several genes in the MAVS signaling pathway were also upregulated by H5N1, which contributed to further amplification of the signal. We also examined hematoxylin-eosin stained tissue sections and observe evidence of lung pathology and splenocyte depletion with both H5N1 viruses at 3 dpi, and recovery by 6 dpi. However, for both H5N1 strains we observed inflammation around neurons in brain, with increased cytokine expression in some individuals. Our findings reveal HPAI H5N1 viruses induced stronger innate immune responses to the infection, while LPAI viruses elicit a milder response.

Development of a DAS-ELISA for detection of H9N2 avian influenza virus

H9N2 avian influenza virus is threatening animals and public health systems. Effective diagnosis is imperative to control the disease. Thus, we developed a panel of monoclonal antibodies (Mabs) against the H9N2 avian influenza virus (AIV) and implemented a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) to detect the H9 viral antigen. Hybridomas 4D10 and 5G2 were screened to secrete immunoglobulin G (IgG) and IgA, respectively. Antibody 4D10 was used as the capture antibodies and HRP labeled 5G2 as the detector antibody. The specificity of the optimized DAS-ELISA was evaluated by using AIV subtypes H1, H3, H5, H9 and H10. Specimens containing AIV H9 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limit of the DAS-ELISA is 10−2.3 TCID50 (50% Tissue culture infective doses). Negative-positive threshold was 0.211 (OD630). In comparison with virus isolation the sensitivity and specificity of DAS-ELISA were found to be 98.9% and 98.1% respectively. Taken together, the newly developed Mab-based DAS-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H9 subtype AIV.

Molecular characterization of H10 subtype avian influenza viruses isolated from poultry in Eastern China.

In recent years, avian-origin H10 influenza viruses have proved capable of infecting human beings, and they pose a potential public health threat. Seven H10 avian influenza viruses (AIVs), H10N3 (n = 2), H10N7 (n = 1), and H10N8 (n = 4), were isolated from chickens in Zhejiang Province, Eastern China, during surveillance of AIVs in live poultry markets in 2016 and 2017. Phylogenetic analysis indicated that Zhejiang H10 strains received gene segments from H10, H3, and H7 viruses from birds in East Asia. Animal inoculation tests showed that these isolates have low pathogenicity in mice and can replicate in this species. Our findings suggest these H10 AIVs have the ability to adapt to chicken or other poultry, and highlight the need of long-term surveillance.

Transmission Dynamics of Highly Pathogenic Avian Influenza Virus A(H5Nx) Clade 2.3.4.4, North America, 2014-2015.

Eurasia highly pathogenic avian influenza virus (HPAIV) H5 clade 2.3.4.4 emerged in North America at the end of 2014 and caused outbreaks affecting >50 million poultry in the United States before eradication in June 2015. We investigated the underlying ecologic and epidemiologic processes associated with this viral spread by performing a comparative genomic study using 268 full-length genome sequences and data from outbreak investigations. Reassortant HPAIV H5N2 circulated in wild birds along the Pacific flyway before several spillover events transmitting the virus to poultry farms. Our analysis suggests that >3 separate introductions of HPAIV H5N2 into Midwest states occurred during March–June 2015; transmission to Midwest poultry farms from Pacific wild birds occurred ≈1.7–2.4 months before detection. Once established in poultry, the virus rapidly spread between turkey and chicken farms in neighboring states. Enhanced biosecurity is required to prevent the introduction and dissemination of HPAIV across the poultry industry.

The Pandemic Threat of Emerging H5 and H7 Avian Influenza Viruses.

The 1918 H1N1 Spanish Influenza pandemic was the most severe pandemic in modern history. Unlike more recent pandemics, most of the 1918 H1N1 virus’ genome was derived directly from an avian influenza virus. Recent avian-origin H5 A/goose/Guangdong/1/1996 (GsGd) and Asian H7N9 viruses have caused several hundred human infections with high mortality rates. While these viruses have not spread beyond infected individuals, if they evolve the ability to transmit efficiently from person-to-person, specifically via the airborne route, they will initiate a pandemic. Therefore, this review examines H5 GsGd and Asian H7N9 viruses that have caused recent zoonotic infections with a focus on viral properties that support airborne transmission. Several GsGd H5 and Asian H7N9 viruses display molecular changes that potentiate transmission and/or exhibit ability for limited transmission between ferrets. However, the hemagglutinin of these viruses is unstable; this likely represents the most significant obstacle to the emergence of a virus capable of efficient airborne transmission. Given the global disease burden of an influenza pandemic, continued surveillance and pandemic preparedness efforts against H5 GsGd and Asian lineage H7N9 viruses are warranted.

Evolution of high pathogenicity of H5 avian influenza virus: haemagglutinin cleavage site selection of reverse-genetics mutants during passage in chickens

Low pathogenicity avian influenza viruses (LPAIVs) are generally asymptomatic in their natural avian hosts. LPAIVs can evolve into highly pathogenic forms, which can affect avian and human populations with devastating consequences. The switch to highly pathogenic avian influenza virus (HPAIV) from LPAIV precursors requires the acquisition of multiple basic amino acids in the haemagglutinin cleavage site (HACS) motif. Through reverse genetics of an H5N1 HPAIV, and experimental infection of chickens, we determined that viruses containing five or more basic amino acids in the HACS motif were preferentially selected over those with three to four basic amino acids, leading to rapid replacement with virus types containing extended HACS motifs. Conversely, viruses harbouring low pathogenicity motifs containing two basic amino acids did not readily evolve to extended forms, suggesting that a single insertion of a basic amino acid into the cleavage site motif of low-pathogenic viruses may lead to escalating selection for extended motifs. Our results may explain why mid-length forms are rarely detected in nature. The stability of the short motif suggests that pathogenicity switching may require specific conditions of intense selection pressure (such as with high host density) to boost selection of the initial mid-length HACS forms.

High frequency of reassortment after co-infection of chickens with the H4N6 and H9N2 influenza A viruses and the biological characteristics of the reassortants

H4 and H9 avian influenza viruses (AIVs) are two of the most prevalent influenza viruses worldwide. The co-existence of H4 and H9 viruses in multiple avian species provides an opportunity for the generation of novel reassortants and for viral evolution. The diversity of the biological characteristics of the reassortants enhances the potential threat to the poultry industry and to public health. To evaluate the reassortment of these viruses and the potential public risk of the reassortants, we co-infected chickens with H4N6 and H9N2 viruses derived from poultry and tested the replication and virulence of the reassortant viruses in mice. A high frequency of reassortment was detected in chickens after co-infection with these two viruses and nine reassortants of six genotypes were purified from the chicken samples. Two H9N2 reassortants containing the PA of the parent H4N6 virus showed higher virulence than the parent H9N2 virus, revealing the significant role of the H4N6 wt virus PA gene in viral reassortment. Analysis of the polymerase activity of the ribonucleoprotein (RNP) complex in vitro suggested that the PA of H4N6 wt origin enhanced polymerase activity. Our results indicate that co-infection of an avian individual with the H4N6 and H9N2 viruses leads to a high frequency of reassortment and generates some reassortants that have higher virulence than the wild-type viruses in mammals. These results highlight the potential public risk of the avian influenza reassortants and the importance of surveillance of the co-existence of the H4N6 and H9N2 viruses in avian species and other animals.

Prevalence of Multiple Subtypes of Avian Influenza Virus Antibodies in Egg Yolks of Mallards ( Anas platyrhynchos) and White-winged Terns ( Chlidonias leucopterus) in the Northeastern Republic of China.

Wild birds are natural hosts of avian influenza viruses (AIV) and can transmit viruses to poultry and other species. To monitor the prevalence of AIV antibodies, 211 eggs from wild Mallards ( Anas platyrhynchos) and 177 from wild White-winged Terns ( Chlidonias leucopterus) were collected from Zhalong Wetland and Xianghai Wetland in northeastern Republic of China from April to September, 2016. A hemagglutinin inhibition test detected the presence of H1, H3, H5, and H7 subtype-specific antibodies. The prevalences of AIV antibodies of subtypes H1 and H3 were relatively high while the prevalences of H5 and H7 AIV subtype antibody were low. In Zhalong Wetland, the prevalence of H1 AIV subtype antibody in Mallards was the highest, with a percentage of 11.0%. Prevalence of all AIV subtype-specific antibodies in Mallards was higher than those in White-winged Terns.

Live bird markets in Bangladesh as a potentially important source for Avian Influenza Virus transmission

Live bird markets (LBM) are important for trading poultry in many developing countries where they are being considered hotspots of Avian Influenza Virus (AIV) prevalence and contamination. An active surveillance for Avian Influenza Virus (AIV) was conducted on four species of LBM birds (chickens, ducks, quails and pigeons) from 10 of the largest LBM in Chittagong, Bangladesh, and two species of peri-domestic wild birds (house crow and Asian pied starling) in their direct vicinity from November 2012 until September 2016. Our aim was to identify the scale and annual pattern of AIV circulation in both the LBM birds and the two per-domestic wild bird species living in close proximity of the LBM. In the latter two species, the annual pattern in AIV antibody prevalence was additionally investigated. A total of 4770 LBM birds and 1119 peri-domestic wild birds were sampled. We used rt-PCR for detection of the AIV M-gene and AIV subtypes H5, H7 and H9 from swab samples. We used c-ELISA for AIV antibody detection from serum samples of peri-domestic wild birds. Average AIV prevalence among the four LBM species varied between 16 and 28%, whereas no AIV was detected in peri-domestic wild birds by rt-PCR. In all LBM species we found significantly higher AIV prevalence in winter compared to summer. A similar pattern was found in AIV antibody prevalence in peri-domestic wild birds feeding in the direct vicinity of LBM. For the subtypes of AIV investigated, we found a significantly higher proportion of AIV H5 in LBM chickens and H9 in LBM ducks. No H7 was detected in any of the investigated samples. We conclude that AIV and notably AIV H5 and H9 were circulating in the investigated LBM of Bangladesh with clear seasonality that matched the prevalence of AIV antibodies of peri-domestic wild birds. These patterns show great resemblance to the annual outbreak patterns in Bangladeshi poultry industry. Our data suggest considerable exchange of AIV within and among the four LBM bird species and peri-domestic wild birds, which likely contributes to the maintenance of the AIV problems in Bangladesh. Increasing biosecurity and notably reducing the direct and indirect mixing of various domestic bird species and peri-domestic wild birds and developing all-in-all-out selling systems with regular use of disinfectant are likely to reduce the risk of transmission and spread of AIV, including HPAI.

Replication and pathogenic potential of influenza A virus subtypes H3, H7, and H15 from free-range ducks in Bangladesh in mammals

Surveillance of wild aquatic birds and free-range domestic ducks in the Tanguar Haor wetlands in Bangladesh has identified influenza virus subtypes H3N6, H7N1, H7N5, H7N9, and H15N9. Molecular characterization of these viruses indicates their contribution to the genesis of new genotypes of H5N1 influenza viruses from clade 2.3.2.1a that are dominant in poultry markets in Bangladesh as well as to the genesis of the highly pathogenic H5N8 virus currently causing disease outbreaks in domestic poultry in Europe and the Middle East. Therefore, we studied the antigenicity, replication, and pathogenicity of influenza viruses isolated from Tanguar Haor in the DBA/2J mouse model. All viruses replicated in the lung without prior mammalian adaptation, and H7N1 and H7N9 viruses caused 100% and 60% mortality, respectively. H7N5 viruses replicated only in the lungs, whereas H7N1 and H7N9 viruses also replicated in the heart, liver, and brain. Replication and transmission studies in mallard ducks showed that H7N1 and H7N9 viruses replicated in ducks without clinical signs of disease and shed at high titers from the cloaca of infected and contact ducks, which could facilitate virus transmission and spread. Our results indicate that H7 avian influenza viruses from free-range ducks can replicate in mammals, cause severe disease, and be efficiently transmitted to contact ducks. Our study highlights the role of free-range ducks in the spread of influenza viruses to other species in live poultry markets and the potential for these viruses to infect and cause disease in mammals.