Murine Model for the Study of Influenza D Virus.

A novel genus within the Orthomyxoviridae family was identified in the United States and named influenza D virus (IDV). Bovines have been proposed to be the primary host, and three main viral lineages (D/OK-like, D/660-like, and D/Japan-like) have been described. Experimental infections had previously been performed in swine, ferrets, calves, and guinea pigs in order to study IDV pathogenesis. We developed a murine experimental model to facilitate the study of IDV pathogenesis and the immune response. DBA/2 mice were inoculated with 105 50% tissue culture infective dose (TCID50) of D/bovine/France/5920/2014 (D/OK-like). No clinical signs or weight loss were observed. Viral replication was observed mainly in the upper respiratory tract (nasal turbinates) but also in the lower respiratory tract of infected mice, with a peak at 4 days postinfection. Moreover, the virus was also detected in the intestines. All infected mice seroconverted by 14 days postinfection. Transcriptomic analyses demonstrated that IDV induced the activation of proinflammatory genes, such as gamma interferon (IFN-γ) and CCL2. Inoculation of NF-κB-luciferase and Ifnar1-/- mice demonstrated that IDV induced mild inflammation and that a type I interferon response was not necessary in IDV clearance. Adaptation of IDV by serial passages in mice was not sufficient to induce disease or increased pathogenesis. Taken together, present data and comparisons with the calf model show that our mouse model allows for the study of IDV replication and fitness (before selected viruses may be inoculated on calves) and also of the immune response.IMPORTANCE Influenza D virus (IDV), a new genus of Orthomyxoviridae family, presents a large host range and a worldwide circulation. The pathogenicity of this virus has been studied in the calf model. The mouse model is frequently used to enable a first assessment of a pathogen's fitness, replication, and pathogenesis for influenza A and B viruses. We showed that DBA/2 mice are a relevant in vivo model for the study of IDV replication. This model will allow for rapid IDV fitness and replication evaluation and will enable phenotypic comparisons between isolated viruses. It will also allow for a better understanding of the immune response induced after IDV infection.

Avian influenza surveillance in Central and West Africa, 2010-2014.

Avian influenza virus (AIV) is an important zoonotic pathogen, resulting in global human morbidity and mortality and substantial economic losses to the poultry industry. Poultry and wild birds have transmitted AIV to humans, most frequently subtypes H5 and H7, but also different strains and subtypes of H6, H9, and H10. Determining which birds are AIV reservoirs can help identify human populations that have a high risk of infection with these viruses due to occupational or recreational exposure to the reservoir species. To assess the prevalence of AIV in tropical birds, from 2010 to 2014, we sampled 40 099 birds at 32 sites in Central Africa (Cameroon, Central African Republic, Congo-Brazzaville, Gabon) and West Africa (Benin, Côte d'Ivoire, Togo). In Central Africa, detection rates by real-time RT-PCR were 16·6% in songbirds (eight passerine families, n = 1257), 16·4% in kingfishers (family Alcedinidae, n = 73), 8·2% in ducks (family Anatidae, n = 564), and 3·65% in chickens (family Phasianidae, n = 1042). Public health authorities should educate human cohorts that have high exposure to these bird populations about AIV and assess their adherence to biosecurity practices, including Cameroonian farmers who raise small backyard flocks.

Avian flu: multiple introductions of H5N1 in Nigeria.

As the avian influenza virus H5N1 swept from Asia across Russia to Europe, Nigeria was the first country in Africa to report the emergence of this highly pathogenic virus. Here we analyse H5N1 sequences in poultry from two different farms in Lagos state and find that three H5N1 lineages were independently introduced through routes that coincide with the flight paths of migratory birds, although independent trade imports cannot be excluded.

Molecular epidemiology of chicken anemia virus (CAV) in southeastern Chinese live birds markets.

Between January 2004 and December 2005, cloacal swabs from essentially healthy chickens and silky chickens from live birds markets in Guangdong and Hunan provinces in southeastern China were screened for chicken anemia virus (CAV) by polymerase chain reaction. Phylogenetic analysis of the major structural protein VP1 sequences showed no clear genotype cluster and no correlation with the geographic origin of CAV strains. Virus evolution at the amino acid level was very slow, which corresponds to a strong negative selection of the VP1 gene in China and worldwide. A high proportion (87%) of birds was CAV positive, suggesting that many farms in the region were infected. Further investigations are necessary to evaluate the economic losses caused by CAV and the cost-benefit of vaccination.

Avian metapneumovirus subtype A in China and subtypes A and B in Nigeria.

In order to detect and characterize avian metapneumovirus, organs or swabs were collected from 697 chicken and 110 turkeys from commercial farms in Southwestern Nigeria and from 107 chickens from live bird markets in Southeastern China. In Nigeria, 15% and 6% of the chicken and turkey samples, respectively, and 39% of the chicken samples from China, were positive for aMPV genome by PCR. The sequence of a 400 nt fragment of the attachment protein gene (G gene) revealed the presence of aMPV subtype A in both Nigeria and Southeastern China. Essentially identical subtype A viruses were found in both countries and were also previously reported from Brazil and the United Kingdom, suggesting a link between these countries or a common source of this subtype. In Nigeria, subtype B was also found, which may be a reflection of chicken importations from most major poultry-producing countries in Europe and Asia. In order to justify countermeasures, further studies are warranted to better understand the metapneumoviruses and their impact on poultry production.

Seroprevalence of avian influenza virus, infectious bronchitis virus, reovirus, avian pneumovirus, infectious laryngotracheitis virus, and avian leukosis virus in Nigerian poultry.

Eight poultry farms in Nigeria, including chickens from nine breeder, 14 broiler, 28 pullet, 11 layer, and three cockerel flocks, were tested for antibody seroprevalence to the following poultry viruses of potential economic importance: infectious bronchitis virus (IBV), avian reovirus, avian pneumovirus (APV), infectious laryngotracheitis virus (ILTV), avian influenza virus (AIV), and avian leukosis virus (ALV). Serum samples were collected between 1999 and 2004 and were tested for antibodies using commercial enzyme-linked immunosorbent assay (ELISA) kits. Seroprevalence was very high for IBV (84%); intermediate for reovirus (41%), APV (40%), and ILTV (20%); and very low for ALV (<5%) antibodies. By commercial ELISA, the seroprevalence of antibodies against AIV was, in some flocks, up to 63%. However, more specific assays did not confirm AIV antibodies, indicating that all flocks tested were free of avian influenza antibodies. Birds seemed to be first infected by IBV (at about 7 wk of age), then by reovirus at 12 wk, before they became infected by APV (week 25) and ILTV (week 30). This is the first report of serological evidence of the above viruses in West Africa. Further studies are necessary to assess economic losses due to these avian viruses and the costs and benefits of countermeasures.

Prevalence of Newcastle disease virus and infectious bronchitis virus in avian influenza negative birds from live bird markets and backyard and commercial farms in Ivory-Coast.

Newcastle disease (ND) and infectious bronchitis (IB) are two major viral diseases affecting the respiratory tracts of birds and whose impact on African poultry is still poorly known. In the present study we aimed at assessing NDV and IBV prevalences in Ivory-Coast by molecular screening of >22,000 avian swabs by nested PCR and by serology testing of close to 2000 avian sera from 2010 through 2012. The NDV and IBV seroprevalences over the study period reached 22% and 72%, respectively. We found 14.7% pooled swabs positive by PCR for NDV and 14.6% for IBV. Both pathogens are therefore endemic in Ivory-Coast. Economic losses associated with NDV and IBV infections still need to be evaluated.

Subpopulations in aMPV vaccines are unlikely to be the only cause of reversion to virulence.

Avian metapneumovirus (aMPV) infects respiratory and reproductive tracts of domestic poultry, often involving secondary infections, and leads to serious economic losses in most parts of the world. While in general disease is effectively controlled by live vaccines, reversion to virulence of those vaccines has been demonstrated on several occasions. Consensus sequence mutations involved in the process have been identified in more than one instance. In one previous subtype A aMPV candidate vaccine study, small subpopulations were implicated. In the current study, the presence of subpopulations in a subtype B vaccine was investigated by deep sequencing. Of the 19 positions where vaccine (strain VCO3/50) and progenitor (strain VCO3/60616) consensus sequences differed, subpopulations were found to have sequence matching progenitor sequence in 4 positions. However none of these mutations occurred in a virulent revertant of that vaccine, thereby demonstrating that the majority progenitor virus population had not survived the attenuation process, hence was not obviously involved in any return to virulence. However within the vaccine, a single nucleotide variation was found which agreed with consensus sequence of a derived virulent revertant virus, hence this and other undetected, potentially virulent subpopulations, can be involved in reversion. Much deeper sequencing of progenitor, vaccine and revertant may clarify whether problematic virulent subpopulations are present and therefore whether these need to be routinely removed during aMPV vaccine preparation prior to registration and release.

Impact of prior seasonal influenza vaccination and infection on pandemic A (H1N1) influenza virus replication in ferrets.

Early epidemiologic and serologic studies have suggested pre-existing immunity to the pandemic A (H1N1) 2009 influenza virus (H1N1pdm) may be altering its morbidity and mortality in humans. To determine the role that contemporary seasonal H1N1 virus infection or trivalent inactivated vaccine (TIV) might be playing in this immunity we conducted a vaccination-challenge study in ferrets. Vaccination with TIV was unable to alter subsequent morbidity or contact transmission in ferrets following challenge with H1N1pdm. Conversely, prior infection with the contemporary seasonal H1N1 strain altered morbidity, but not transmission, of H1N1pdm despite the detection of only minimal levels of cross reactive antibodies.

Genotyping assay for the identification of 2009-2010 pandemic and seasonal H1N1 influenza virus reassortants.

New Zealand identified its first pandemic H1N1 influenza cases in late April 2009, immediately prior to the historical start of the New Zealand influenza season. Both pandemic and oseltamivir-resistant seasonal H1N1 viruses cocirculated in the population for a period of time. Thus, concerns were raised about the possibility of reassortment events between the two strains. An RT-PCR-based genotyping assay was developed so that H1N1 influenza coinfections and reassortants could be detected quickly. The assay differentiated effectively the seasonal and pandemic strains. It also confirmed the identification of the first reported coinfection of pandemic and seasonal H1N1 strains during the 2009 Southern Hemisphere influenza season in New Zealand.

Animal influenza epidemiology.

Influenza A viruses exist within their natural host, aquatic birds, in a number of antigenic subtypes. Only a few of these subtypes have successfully crossed into other avian and mammalian hosts. This brief review will focus on just three examples of viruses that have successfully passed between species; avian H5NI1 and H9N2 viruses and H3N2 viruses which have transmitted from aquatic birds to humans and then to swine. Although there are a number of other subtypes that have also transmitted successfully between species, these three selected examples have spread and evolved in different ways, exemplifying the complexity of influenza A virus epidemiology.

Molecular and antigenic evolution and geographical spread of H5N1 highly pathogenic avian influenza viruses in western Africa.

In Africa, highly pathogenic avian influenza H5N1 virus was first detected in northern Nigeria and later also in other regions of the country. Since then, seven other African countries have reported H5N1 infections. This study reports a comparison of full-length genomic sequences of H5N1 isolates from seven chicken farms in Nigeria and chicken and hooded vultures in Burkina Faso with earlier H5N1 outbreaks worldwide. In addition, the antigenicity of Nigerian H5N1 isolates was compared with earlier strains. All African strains clustered within three sublineages denominated A (south-west Nigeria, Niger), B (south-west Nigeria, Egypt, Djibouti) and C (northern Nigeria, Burkina Faso, Sudan, Côte d'Ivoire), with distinct nucleotide and amino acid signatures and distinct geographical distributions within Africa. Probable non-African ancestors within the west Asian/Russian/European lineage distinct from the south-east Asian lineages were identified for each sublineage. All reported human cases in Africa were caused by sublineage B. Substitution rates were calculated on the basis of sequences from 11 strains from a single farm in south-west Nigeria. As H5N1 emerged essentially at the same time in the north and south-west of Nigeria, the substitution rates confirmed that the virus probably did not spread from the north to the south, given the observed sequence diversity, but that it entered the country via three independent introductions. The strains from Burkina Faso seemed to originate from northern Nigeria. At least two of the sublineages also circulated in Europe in 2006 as seen in Germany, further suggesting that the sublineages had already emerged outside of Africa and seemed to have followed the east African/west Asian and Black Sea/Mediterranean flyways of migratory birds.

Molecular epidemiology of chicken anemia virus in Nigeria.

Between February 2002 and May 2004, chicken anemia virus (CAV) was detected by PCR in organ samples from 14 flocks of poultry farms in Lagos, Ogun and Oyo States in Southwestern Nigeria. The farms reported low (<5%) to high mortalities (up to 100%) with various lesions at necropsy. The complete VP1 gene of 30 of these positive strains was sequenced. Strains that diverged by up to 4.4% on a nucleotide level differed only by up to 2.5% at the amino acid level (7 aa) as a result of clustered silent mutations. No amino acid substitutions specific for Nigerian strains were observed. Some birds had a CAV mixed infection. Genetic clustering of the VP1 gene did not correlate with differences in flock mortality but the co-infection of CAV with IBDV may be particularly lethal. This first molecular epidemiological study of CAV in Africa shows that the Nigerian strains cluster with viruses from very diverse geographic origins and were almost as diverse (4.4%) as all other strains combined (5.8%).