Jung Yum

Highly pathogenic avian influenza A(H5N8) virus from waterfowl, South Korea, 2014.

To the Editor: To date, 18 hemagglutinin (HA) subtypes and 11 neuraminidase (NA) subtypes have been identified in influenza A viruses (1–4). Influenza A viruses containing HA subtypes 1–16 circulate in aquatic birds (1,2), whereas those harboring HA subtypes 17 and 18 are found in bats (3,4). n nOn January 18, 2014, the government of South Korea reported an outbreak of highly pathogenic avian influenza A(H5N8) virus in breeding ducks in the southern part of Jeollabuk-Do Province (5). More than 12 million poultry have since been culled, but the spread of the virus continues in duck and chicken farms. We report the genetic characterization of this virus. n nOn February 15, 2014, a total of 200 fecal samples were collected from waterfowl in the Pungse River in Chungnam Province, which is geographically close to Jeollabuk-Do Province. All samples were inoculated into hens’ eggs, and influenza A viruses were confirmed by PCR by using influenza A–specific nucleoprotein (NP) primers. We obtained 1 isolate, A/waterfowl/Korea/S005/2014 (H5N8), and sequenced the full regions of all 8 genes as described (6). These sequences were deposited into GenBank under accession nos. {type:entrez-nucleotide-range,attrs:{text:KJ511809-KJ511816,start_term:KJ511809,end_term:KJ511816,start_term_id:629688568,end_term_id:629688575}}KJ511809-KJ511816. n nWe conducted a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi, http://platform.gisaid.org/epi3/frontend#4ead5c) to identify the closest gene sequences to those of A/waterfowl/Korea/S005/2014 (H5N8) (Table). Sequences for polymerase basic (PB) 2 (99% homology), HA (97% homology), and NP (99% homology) genes were closely related to those of A/wild duck/Shandong/628/2011 (H5N1). Sequences for PB1 (99% homology), polymerase acidic subunit (PA) (98% homology), matrix (M) (99% homology), and nonstructural (NS) (99% homology) genes were closely related to those of A/duck/Jiangsu/1-15/2011 (H4N2). Sequences for the NA (98% homology) gene were closely related to that of A/duck/Jiangsu/k1203/2010 (H5N8). Phylogenic analysis showed that all 8 genes of A/waterfowl/Korea/S005/2014 (H5N8) belonged to the Eurasian lineage, and that the HA gene clustered with clade 2.3.4 (Technical Appendix Figure 1). n n n nTable n nNucleotide homology of genes of influenza virus strain A/waterfowl/Korea/S005/2014 (H5N8) to the closest related influenza virus strains* n n n nWe further analyzed the amino acid sequences of the virus isolate (online Technical Appendix Table 1). Positions 138 and 160 of the HA protein (H3 numbering) contained an alanine (A) residue, which was previously found to be related to enhanced binding to the human influenza receptor (7). The connecting peptide of HA contained an insertion of 4 basic amino acids (arginine-arginine-arginine-lysine), which is the same as in the HA of A/duck/Korea/Buan2/2014 (H5N8), an isolate from a duck farm in South Korea (GenBank accession no. {type:entrez-nucleotide-range,attrs:{text:KJ413839.1-KJ413846.1,start_term:KJ413839.1,end_term:KJ413846.1,start_term_id:589826076,end_term_id:589826093}}KJ413839.1-KJ413846.1). Aspartic acid was found in M1 at position 30 and alanine at position 215; this combination has been connected with increased virulence in mice (8). The NS1 sequence contained serine at position 42, which is related to the enhanced pathogenicity in mice, but a truncation of the amino acids at positions 218–230 that has been linked with reduced pathogenicity in mice (9) was not identified. Asparagine was identified at position 31 of M2, which is the same in M2 of A/duck/Korea/Buan2/2014 (H5N8) and confers resistance to amantadine and rimantadine (10). n nBecause all 8 genes of A/waterfowl/Korea/S005/2014 (H5N8) are closely related to those of the A/duck/Korea/Buan2/2014 (H5N8) isolate that was obtained from a duck farm, it is likely that A/waterfowl/Korea/S005/2014 (H5N8) originated from infected waterfowl that had visited poultry on an infected farm (Technical Appendix Figure 1). Our laboratory has studied the feces of wild birds in Chungnam Province since 2009, surveying >20,000 fecal samples from wild birds in this area each year, but we had not previously isolated avian influenza A(H5N8) virus from any samples. n nThe genetic analysis of the A/waterfowl/Korea/S005/2014 (H5N8) isolate indicates that this novel strain may have been created by the reassortment of PB2, HA, and NP segments from H5N1-like avian influenza virus; PB1, PA, M, and NS segments from H4N2-like avian influenza virus; and NA segments from H5N8-like avian influenza virus (Technical Appendix Figure 2). Most genes of the virus we isolated are related to those of avian influenza viruses isolated in China, but the HA gene of A/waterfowl/Korea/S005/2014 (H5N8) showed only 97% homology to the closest HA gene in GenBank, which indicates that this gene may have been created in poultry in South Korea. To our knowledge, no outbreak of this virus in poultry farms in China has been reported, and we found no previous reports in the literature that migratory birds could carry the virus. Taken together, our data suggest that A/waterfowl/Korea/S005/2014 (H5N8) may have been reassorted in a duck farm in South Korea. n nTechnical Appendix: nPhylogenetic analysis, schematic diagram, and identification of amino acids of influenza virus strain A/waterfowl/Korea/S005/2014 (H5N8). n nClick here to view.(1.2M, pdf)

Transmissibility of novel H7N9 and H9N2 avian influenza viruses between chickens and ferrets

Previous studies have shown that the H7N9 avian influenza virus cannot be transmitted efficiently between ferrets via respiratory droplets. Here, we studied the infectivity of the H7N9 avian influenza virus in chickens and its transmissibility from infected to naïve chickens and ferrets. The H7N9 virus (A/Anhui/1/2013) replicated poorly in chickens and could not be transmitted efficiently from infected chickens to naïve chickens and ferrets. H7N9 virus was shed from chicken tracheae for only 2 days after infection and from chicken cloacae for only 1 day after infection, while the H9N2 avian influenza virus, which is endemic in chickens in many Asian countries, was shed from tracheae and cloacae for 8 days after infection. Taken together, our results suggest that chickens may be a poor agent of transmission for the H7N9 virus to other chickens and to mammals, including humans.

The severe pathogenicity of alveolar macrophage-depleted ferrets infected with 2009 pandemic H1N1 influenza virus.

The in vivo role of alveolar macrophages in the infections with 2009 pandemic H1N1 influenza virus is not as yet known. Ferret study shows that alveolar macrophages are critical for lowering the risk of severe outcomes in 2009 pandemic H1N1 influenza virus infections. Up to 40% of the infected ferrets depleted of alveolar macrophages died, with elevated body temperature and major loss of body weight in contrast to infected ferrets not depleted of alveolar macrophages. The higher viral titers in the lungs were detected in infected ferrets depleted of alveolar macrophages than infected ferrets not depleted of alveolar macrophages 5 days after infection. The inflammatory chemokines were induced at greater levels in the lungs of infected ferrets depleted of alveolar macrophages than in those of infected ferrets not depleted of alveolar macrophages. Our study implies that alveolar macrophages are important for controlling the infections of 2009 pandemic H1N1 influenza virus.

H3N2 canine influenza virus causes severe morbidity in dogs with induction of genes related to inflammation and apoptosis

Dogs are companion animals that live in close proximity with humans. Canine H3N2 influenza virus has been isolated from pet dogs that showed severe respiratory signs and other clinical symptoms such as fever, reduced body weight, and interstitial pneumonia. The canine H3N2 influenza virus can be highly transmissible among dogs via aerosols. When we analyzed global gene expression in the lungs of infected dogs, the genes associated with the immune response and cell death were greatly elevated. Taken together, our results suggest that canine H3N2 influenza virus can be easily transmitted among dogs, and that severe pneumonia in the infected dogs may be partially due to the elevated expression of genes related to inflammation and apoptosis.

Severe pathogenesis of influenza B virus in pregnant mice

The study on pathogenesis of influenza B virus during pregnancy is limited. Here, we showed using a mouse model that influenza B virus could cause severe disease including death during pregnancy. Infected pregnant mice resulted in 40% mortality, but infected age-matched non-pregnant mice did not show any death. Infected pregnant mice contained high viral loads in lungs with the elevated inductions of inflammatory cytokines and chemokines than infected non-pregnant mice. Infected pregnant mice delivered lower number of neonates than uninfected pregnant mice, suggesting adverse effects of influenza B virus on fetuses. Progesterone which is important for maintaining pregnancy was reduced in uteruses of infected pregnant mice than in those of uninfected pregnant mice. Taken together, our results suggest that influenza B virus can cause severe disease during pregnancy, and that preventive measures including vaccination may be important for protecting women during pregnancy.

Red Ginseng-containing diet helps to protect mice and ferrets from the lethal infection by highly pathogenic H5N1 influenza virus

The highly pathogenic (HP) H5N1 influenza virus is endemic in many countries and has a great potential for a pandemic in humans. The immune-enhancing prowess of ginseng has been known for millennia. We aimed to study whether mice and ferrets fed with Red Ginseng could be better protected from the lethal infections of HP H5N1 influenza virus than the infected unfed mice and ferrets. We fed mice and ferrets with Red Ginseng prior to when they were infected with HP H5N1 influenza virus. The mice and ferrets fed with a 60-day diet containing Red Ginseng could be protected from lethal infections by HP H5N1 influenza virus (survival rate of up to 45% and 40%, respectively). Interferon-α and -γ antiviral cytokines were significantly induced in the lungs of mice fed Red Ginseng, compared to mice fed an unsupplemented diet. These data suggest that the diet with the immune-enhancing Red Ginseng could help humans to overcome the infections by HP H5N1 influenza virus.

H7N9 Influenza Virus Is More Virulent in Ferrets than 2009 Pandemic H1N1 Influenza Virus

The novel H7N9 influenza virus has been infecting humans in China since February 2013 and with a mortality rate of about 40%. This study compared the pathogenicity of the H7N9 and 2009 pandemic H1N1 influenza viruses in a ferret model, which shows similar symptoms to those of humans infected with influenza viruses. The H7N9 influenza virus caused a more severe disease than did the 2009 pandemic H1N1 influenza virus. All of the ferrets infected with the H7N9 influenza virus had died by 6 days after infection, while none of those infected with the 2009 pandemic H1N1 influenza virus died. Ferrets infected with the H7N9 influenza virus had higher viral titers in their lungs than did those infected with the 2009 pandemic H1N1 influenza virus. Histological findings indicated that hemorrhagic pneumonia was caused by infection with the H7N9 influenza virus, but not with the 2009 pandemic H1N1 influenza virus. In addition, the lung tissues of ferrets infected with the H7N9 influenza virus contained higher levels of chemokines than did those of ferrets infected with the 2009 pandemic H1N1 influenza virus. This study suggests that close monitoring is needed to prevent human infection by the lethal H7N9 influenza virus.

Greater virulence of highly pathogenic H5N1 influenza virus in cats than in dogs

Highly pathogenic H5N1 influenza virus continues to infect animals and humans. We compared the infectivity and pathogenesis of H5N1 virus in domestic cats and dogs to find out which animal is more susceptible to H5N1 influenza virus. When cats and dogs were infected with the H5N1 virus, cats suffered from severe outcomes including death, whereas dogs did not show any mortality. Viruses were shed in the nose and rectum of cats and in the nose of dogs. Viruses were detected in brain, lung, kidney, intestine, liver, and serum in the infected cats, but only in the lung in the infected dogs. Genes encoding inflammatory cytokines and chemokines, Toll-like receptors, and apoptotic factors were more highly expressed in the lungs of cats than in those of dogs. Our results suggest that the intensive monitoring of dogs is necessary to prevent human infection by H5N1 influenza virus, since infected dogs may not show clear clinical signs, in contrast to infected cats.

Protective efficacy of a single dose of baculovirus hemagglutinin-based vaccine in chickens and ducks against homologous and heterologous H5N1 virus infections.

Outbreaks of the highly pathogenic H5N1 virus in poultry and humans are ongoing. Vaccination is an efficient method for prevention of H5N1 infection. Using chickens and ducks, we assessed the efficacy of a vaccine comprising H5N1 hemagglutinin (HA) protein produced in a baculovirus expression system. The immunized chickens and ducks were protected against lethal infection by H5N1 in an antigen dose-dependent manner. Complete protection against homologous challenge and partial protection against heterologous challenge were achieved in chickens immunized with 5u2009μg HA protein and in ducks immunized with 10u2009μg HA protein. The IgG antibody subtype was mainly detected in the sera and tissues, including the lungs. The neuraminidase (NA) inhibition assay was negative in immunized chickens and ducks. Our results indicated that the expressed HA protein by baculovirus was immunogenic to both chickens and ducks, and the immunized chickens and ducks were protected from the lethal infections of highly pathogenic H5N1 influenza virus, though ducks required more HA protein than chickens to be protected. Also, baculovirus HA-vaccinated poultry can be differentiated from infected poultry by NA inhibition assay.

Low infectivity of a novel avian-origin H7N9 influenza virus in pigs

We studied the pathogenesis and transmissibility of a novel avian-origin H7N9 influenza virus in pigs. When pigs were infected with H7N9 influenza virus, they did not show any clear clinical signs (such as sneezing, fever and loss of body weight), and they shed viruses through their noses for 2xa0days after infection. No transmission occurred between infected and naïve pigs. Pigs suffered from mild pneumonia, which was accompanied by the induction of inflammatory cytokines and chemokines such as IL-8 and CCL1. Taken together, our results suggest that pigs may not play an active role in transmitting H7N9 influenza virus to mammals.