Chengjun Li

H7N9 Influenza Viruses Are Transmissible in Ferrets by Respiratory Droplet

H7N9 Adaptation Puzzling and alarming reports of an outbreak in early 2013 of human infections by a low-pathogenicity avian influenza virus has rocked the poultry industry in central eastern China and brought fears of initiating a human pandemic. Over 130 human cases have been reported with 37 deaths until closure of poultry markets accompanied a near-cessation of human case reports. From surveillance sampling of >10,000 isolates obtained during April 2013, Zhang et al. (p. 410, published online 18 July) took 37 isolates of avian origin H7N9 and compared them to human H7N9 isolates. The majority of H7N9 isolates came from live poultry markets, although some originated in pigeons. Sequence analysis indicated that the chicken isolates had retained the avian characteristics at sites on the influenza genes for PB2 and the surface hemagglutinin HA, where adaptive mutations have been observed before. Sequence analysis also showed a higher variability in the internal genes than in HA and neuraminidase NA. By using glycan arrays, it was shown that avian and human isolates bound to human, but also to some extent to avian, receptors. As expected, the virus replicated well in chickens without causing disease, whereas in mice only the human isolates were highly pathogenic. The human virus, but not the avian, transmitted between ferrets through the air. A large survey of H7N9 influenza viruses in China reveals a spectrum of mutation and virulence. A newly emerged H7N9 virus has caused 132 human infections with 37 deaths in China since 18 February 2013. Control measures in H7N9 virus–positive live poultry markets have reduced the number of infections; however, the character of the virus, including its pandemic potential, remains largely unknown. We systematically analyzed H7N9 viruses isolated from birds and humans. The viruses were genetically closely related and bound to human airway receptors; some also maintained the ability to bind to avian airway receptors. The viruses isolated from birds were nonpathogenic in chickens, ducks, and mice; however, the viruses isolated from humans caused up to 30% body weight loss in mice. Most importantly, one virus isolated from humans was highly transmissible in ferrets by respiratory droplet. Our findings indicate nothing to reduce the concern that these viruses can transmit between humans.

H6 influenza viruses pose a potential threat to human health

ABSTRACT Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in a turkey in Massachusetts in 1965. Since 1997, H6 viruses with different neuraminidase (NA) subtypes have been detected frequently in the live poultry markets of southern China. Although sequence information has been gathered over the last few years, the H6 viruses have not been fully biologically characterized. To investigate the potential risk posed by H6 viruses to humans, here we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China from 2008 to 2011. Among the 257 H6 strains tested, 87 viruses recognized the human type receptor. Genome sequence analysis of 38 representative H6 viruses revealed 30 different genotypes, indicating that these viruses are actively circulating and reassorting in nature. Thirty-seven of 38 viruses tested in mice replicated efficiently in the lungs and some caused mild disease; none, however, were lethal. We also tested the direct contact transmission of 10 H6 viruses in guinea pigs and found that 5 viruses did not transmit to the contact animals, 3 viruses transmitted to one of the three contact animals, and 2 viruses transmitted to all three contact animals. Our study demonstrates that the H6 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H6 influenza viruses circulating in nature. IMPORTANCE Avian influenza viruses continue to present a challenge to human health. Research and pandemic preparedness have largely focused on the H5 and H7 subtype influenza viruses in recent years. Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in the United States in 1965. Since 1997, H6 viruses have been detected frequently in the live poultry markets of southern China; however, the biological characterization of these viruses is very limited. Here, we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China and found that 34% of the viruses are able to bind human type receptors and that some of them are able to transmit efficiently to contact animals. Our study demonstrates that the H6 viruses pose a clear threat to human health.

Complex reassortment of multiple subtypes of avian influenza viruses in domestic ducks at the Dongting Lake region of China

ABSTRACT To gain insight into the ecology of avian influenza viruses (AIV), we conducted active influenza virus surveillance in domestic ducks on farms located on the flyway of migratory birds in the Dongting Lake region of Hunan Province, China, from winter 2011 until spring 2012. Specimens comprising 3,030 duck swab samples and 1,010 environmental samples were collected from 101 duck farms. We isolated AIV of various HA subtypes, including H3, H4, H5, H6, H9, H10, H11, and H12. We sequenced the entire coding sequences of the genomes of 28 representative isolates constituting 13 specific subtypes. When the phylogenetic relationships among these isolates were examined, we observed that extensive reassortment events had occurred. Among the 28 Dongting Lake viruses, 21 genotypes involving the six internal genes were identified. Furthermore, we identified viruses or viral genes introduced from other countries, viral gene segments of unknown origin, and a novel HA/NA combination. Our findings emphasize the importance of farmed domestic ducks in the Dongting Lake region to the genesis and evolution of AIV and highlight the need for continued surveillance of domestic ducks in this region.

Avian influenza vaccines against H5N1 'bird flu'.

H5N1 avian influenza viruses (AIVs) have spread widely to more than 60 countries spanning three continents. To control the disease, vaccination of poultry is implemented in many of the affected countries, especially in those where H5N1 viruses have become enzootic in poultry and wild birds. Recently, considerable progress has been made toward the development of novel avian influenza (AI) vaccines, especially recombinant virus vector vaccines and DNA vaccines. Here, we will discuss the recent advances in vaccine development and use against H5N1 AIV in poultry. Understanding the properties of the available, novel vaccines will allow for the establishment of rational vaccination protocols, which in turn will help the effective control and prevention of H5N1 AI.

Prevalence, genetics, and transmissibility in ferrets of Eurasian avian-like H1N1 swine influenza viruses

Significance Animal influenza viruses continue to challenge human public health; surveillance of these viruses and evaluation of their pandemic potential are important for future influenza pandemic preparedness. Pigs are intermediate hosts for novel influenza generation and transfer to humans. Two lineages of H1N1 swine influenza viruses (SIVs), classical H1N1 SIVs and Eurasian avian-like H1N1 (EAH1N1) SIVs, have been circulating in pigs since 1918 and 1979, respectively. The classical H1N1 SIVs emerged in humans as a reassortant and caused the 2009 H1N1 influenza pandemic. Here, we found that, after long-term evolution in pigs, the EAH1N1 SIVs have obtained the traits to cause a human influenza pandemic. Pigs are important intermediate hosts for generating novel influenza viruses. The Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses (SIVs) have circulated in pigs since 1979, and human cases associated with EAH1N1 SIVs have been reported in several countries. However, the biologic properties of EAH1N1 SIVs are largely unknown. Here, we performed extensive influenza surveillance in pigs in China and isolated 228 influenza viruses from 36,417 pigs. We found that 139 of the 228 strains from pigs in 10 provinces in China belong to the EAH1N1 lineage. These viruses formed five genotypes, with two distinct antigenic groups, represented by A/swine/Guangxi/18/2011 and A/swine/Guangdong/104/2013, both of which are antigenically and genetically distinct from the current human H1N1 viruses. Importantly, the EAH1N1 SIVs preferentially bound to human-type receptors, and 9 of the 10 tested viruses transmitted in ferrets by respiratory droplet. We found that 3.6% of children (≤10 y old), 0% of adults, and 13.4% of elderly adults (≥60 y old) had neutralization antibodies (titers ≥40 in children and ≥80 in adults) against the EAH1N1 A/swine/Guangxi/18/2011 virus, but none of them had such neutralization antibodies against the EAH1N1 A/swine/Guangdong/104/2013 virus. Our study shows the potential of EAH1N1 SIVs to transmit efficiently in humans and suggests that immediate action is needed to prevent the efficient transmission of EAH1N1 SIVs to humans.

Recombinant fowlpox virus vector-based vaccine completely protects chickens from H5N1 avian influenza virus.

With the widespread presence of influenza virus H5N1 in poultry and wildlife species, particularly migrating birds, vaccination has become an important control strategy for avian influenza (AI). In this study, the immune efficacy and hemagglutination inhibition (HI) antibody responses induced by a recombinant fowlpox virus (FPV) vector-based rFPV-HA-NA vaccine was evaluated in SPF and commercial chickens. Four-week old SPF chickens vaccinated with one dose of vaccine containing 2 x 10(3) plaque forming units (PFU) of virus were completely protected from H5N1 AI virus 1 week after vaccination, and protective immunity lasted for at least 40 weeks. Two-week old commercial layer chickens were vaccinated with the rFPV-HA-NA vaccine and boosted with the same dose of vaccine following an interval of 18 weeks. The HI antibody titers higher than 4log2 lasted for 52 weeks after the booster immunization. We also examined the efficacy of the rFPV-HA-NA vaccine in SPF chickens administrated by different routes. The results showed that effective application of rFPV-HA-NA vaccine in poultry may be restricted to wing-web puncture, intramuscular or subcutaneous injection. These results demonstrate that the rFPV-HA-NA vaccine is effective in the prevention of infection of H5N1 AI virus.

Single-amino-acid mutation in the HA alters the recognition of H9N2 influenza virus by a monoclonal antibody

We explored the molecular basis of antigenic variation by comparing two H9N2 subtype avian influenza viruses, A/Chicken/Shandong/6/96 (CK/SD/6) and A/Chicken/Guangxi/10/99 (CK/GX/10), that react differently to a monoclonal antibody C/B3. To assess the genetic basis for this antigenic difference, we used reverse genetics to generate a series of chimera and mutants of these two viruses. We found that a single-amino-acid substitution of asparagine for serine at position 145 (S145N) in the HA protein prevents the reaction of CK/SD/6 virus with C/B3. Substitution of serine for asparagine at the same position (N145S) enables the CK/GX/10 to react with C/B3 in hemaglutinin inhibition, immunofluorescence and neutralization assays. We further demonstrated that the amino acid N145 in the H9 HA protein is glycosylated. Our results provide experimental evidence that the glycosylation of HA oligosaccharide attachment sites implicated in antibody binding could have a role in antigenic variation.

Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China

ABSTRACT H4 avian influenza virus (AIV) is one of the most prevalent influenza virus subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that these viruses are undergoing complex and frequent reassortment events. All 32 viruses tested could replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that the H4 AIVs bound to α-2,6-linked glycans, although they retained the binding preference for α-2,3-linked glycans. When we tested the direct-contact transmission of 10 H4 viruses in guinea pigs, we found that three viruses did not transmit to any of the contact animals, one virus transmitted to one of three contact animals, and six viruses transmitted to all three contact animals. When we further tested the respiratory droplet transmissibility of four of the viruses that transmitted efficiently via direct contact, we found that three of them could transmit to one or two of the five exposed animals. Our study demonstrates that the current circulating H4 AIVs can infect, replicate in, and transmit to mammalian hosts, thereby posing a potential threat to human health. These findings emphasize the continual need for enhanced surveillance of H4 AIVs. IMPORTANCE Numerous surveillance studies have documented the wide distribution of H4 AIVs throughout the world, yet the biological properties of H4 viruses have not been well studied. In this study, we found that multiple genotypes of H4 viruses are cocirculating in the live poultry markets of China and that H4 viruses can replicate in mice, possess human-type receptor binding specificity, and transmit between guinea pigs via direct contact. Strikingly, some H4 strains also can transmit via respiratory droplet, albeit with limited efficiency. These results clearly show the potential threat posed by H4 viruses to public health.

H5N1 influenza marker vaccine for serological differentiation between vaccinated and infected chickens

Using plasmid-based reverse genetics, we generated a molecularly altered virus, H5N1/PR8-5B19, containing modified HA and NA genes from A/Goose/Guangdong/1/96 (GS/GD/1/96). In the H5N1/PR8-5B19 virus, the HA cleavage site was modified to resemble that of low-pathogenic avian strains and a portion of the NA stalk region was replaced by the immunodominant 5B19 epitope of the S2 glycoprotein of murine hepatitis virus (MHV). H5N1/PR8-5B19 is not lethal to embryonated eggs or chickens. Chickens immunized with the H5N1/PR8-5B19 inactivated vaccine produced high levels of HI antibody and a measurable antibody response against the MHV 5B19 epitope, and were fully protected against subsequent challenge with different highly pathogenic H5N1 avian influenza viruses. H5N1/PR8-5B19 is therefore an attractive marker vaccine candidate, eliciting a strong, protective antibody response and enabling serological discrimination between vaccinated and wild-type virus-infected chickens.

Identification of PB2 Mutations Responsible for the Efficient Replication of H5N1 Influenza Viruses in Human Lung Epithelial Cells

ABSTRACT Highly pathogenic H5N1 avian influenza viruses have caused outbreaks among poultry worldwide, resulting in sporadic infections in humans with approximately 60% mortality. However, efficient transmission of H5N1 viruses among humans has yet to occur, suggesting that further adaptation of H5N1 viruses to humans is required for their efficient transmission among humans. The viral determinants for efficient replication in humans are currently poorly understood. Here, we report that the polymerase PB2 protein of an H5N1 influenza virus isolated from a human in Vietnam (A/Vietnam/UT36285/2010, virus 36285) increased the growth ability of an avian H5N1 virus (A/wild bird/Anhui/82/2005, virus Wb/AH82) in human lung epithelial A549 cells (however, the reassortant virus did not replicate more efficiently than human 36285 virus). Furthermore, we demonstrate that the amino acid residues at positions 249, 309, and 339 of the PB2 protein from this human isolate were responsible for its efficient replication in A549 cells. PB2 residues 249G and 339M, which are found in the human H5N1 virus, are rare in H5N1 viruses from both human and avian sources. Interestingly, PB2-249G is found in over 30% of human seasonal H3N2 viruses, which suggests that H5N1 viruses may replicate well in human cells when they acquire this mutation. Our data are of value to H5N1 virus surveillance. IMPORTANCE Highly pathogenic H5N1 avian influenza viruses must acquire mutations to overcome the species barrier between avian species and humans. When H5N1 viruses replicate in human respiratory cells, they can acquire amino acid mutations that allow them to adapt to humans through continuous selective pressure. Several amino acid mutations have been shown to be advantageous for virus adaptation to mammalian hosts. Here, we found that amino acid changes at positions 249, 309, and 339 of PB2 contribute to efficient replication of avian H5N1 viruses in human lung cells. These findings are beneficial for evaluating the pandemic risk of circulating avian viruses and for further functional analysis of PB2.