Novel H7N9 Influenza Virus Shows Low Infectious Dose,High Growth Rate,and Efficient Contact Transmission in the Guinea Pig Model

The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.     

Oseltamivir-resistant influenza A viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol

Influenza viruses resistant to the neuraminidase (NA) inhibitor oseltamivir arise under drug selection pressure both in vitro and in vivo. Several mutations in the active site of the viral NA are known to confer relative resistance to oseltamivir, and influenza viruses with certain oseltamivir resistance mutations have been shown to transmit efficiently among cocaged ferrets. However, it is not known whether NA mutations alter aerosol transmission of drug-resistant influenza virus. Here, we demonstrate that recombinant human influenza A/H3N2 viruses without and with oseltamivir resistance mutations (in which NA carries the mutation E119V or the double mutations E119V I222V) have similar in ovo growth kinetics and infectivity in guinea pigs. These viruses also transmit efficiently by the contact route among cocaged guinea pigs, as in the ferret model. However, in an aerosol transmission model, in which guinea pigs are caged separately, the oseltamivir-resistant viruses transmit poorly or not at all; in contrast, the oseltamivir-sensitive virus transmits efficiently even in the absence of direct contact. The present results suggest that oseltamivir resistance mutations reduce aerosol transmission of influenza virus, which could have implications for public health measures taken in the event of an influenza pandemic.     

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.     

Guinea pig model for evaluating the potential public health risk of swine and avian influenza viruses

Background

The influenza viruses circulating in animals sporadically transmit to humans and pose pandemic threats. Animal models to evaluate the potential public health risk potential of these viruses are needed.

Methodology/Principal Findings

We investigated the guinea pig as a mammalian model for the study of the replication and transmission characteristics of selected swine H1N1, H1N2, H3N2 and avian H9N2 influenza viruses, compared to those of pandemic (H1N1) 2009 and seasonal human H1N1, H3N2 influenza viruses. The swine and avian influenza viruses investigated were restricted to the respiratory system of guinea pigs and shed at high titers in nasal tracts without prior adaptation, similar to human strains. None of the swine and avian influenza viruses showed transmissibility among guinea pigs; in contrast, pandemic (H1N1) 2009 virus transmitted from infected guinea pigs to all animals and seasonal human influenza viruses could also horizontally transmit in guinea pigs. The analysis of the receptor distribution in the guinea pig respiratory tissues by lectin histochemistry indicated that both SAα2,3-Gal and SAα2,6-Gal receptors widely presented in the nasal tract and the trachea, while SAα2,3-Gal receptor was the main receptor in the lung.

Conclusions/Significance

We propose that the guinea pig could serve as a useful mammalian model to evaluate the potential public health threat of swine and avian influenza viruses.     

Lethal H5N1 influenza viruses escape host anti-viral cytokine responses

The H5N1 influenza viruses transmitted to humans in 1997 were highly virulent, but the mechanism of their virulence in humans is largely unknown. Here we show that lethal H5N1 influenza viruses, unlike other human, avian and swine influenza viruses, are resistant to the antiviral effects of interferons and tumor necrosis factor alpha. The nonstructural (NS) gene of H5N1 viruses is associated with this resistance. Pigs infected with recombinant human H1N1 influenza virus that carried the H5N1 NS gene experienced significantly greater and more prolonged viremia, fever and weight loss than did pigs infected with wild-type human H1N1 influenza virus. These effects required the presence of glutamic acid at position 92 of the NS1 molecule. These findings may explain the mechanism of the high virulence of H5N1 influenza viruses in humans.     

Key Molecular Factors in Hemagglutinin and PB2 Contribute to Efficient Transmission of the 2009 H1N1 Pandemic Influenza Virus

Animal influenza viruses pose a clear threat to public health. Transmissibility among humans is a prerequisite for a novel influenza virus to cause a human pandemic. A novel reassortant swine influenza virus acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. However, the molecular aspects of influenza virus transmission remain poorly understood. Here, we show that an amino acid in hemagglutinin (HA) is important for the 2009 H1N1 influenza pandemic virus (2009/H1N1) to bind to human virus receptors and confer respiratory droplet transmissibility in mammals. We found that the change from glutamine (Q) to arginine (R) at position 226 of HA, which causes a switch in receptor-binding preference from human α-2,6 to avian α-2,3 sialic acid, resulted in a virus incapable of respiratory droplet transmission in guinea pigs and reduced the virus's ability to replicate in the lungs of ferrets. The change from alanine (A) to threonine (T) at position 271 of PB2 also abolished the virus's respiratory droplet transmission in guinea pigs, and this mutation, together with the HA Q226R mutation, abolished the virus's respiratory droplet transmission in ferrets. Furthermore, we found that amino acid 271A of PB2 plays a key role in virus acquisition of the mutation at position 226 of HA that confers human receptor recognition. Our results highlight the importance of both the PB2 and HA genes on the adaptation and transmission of influenza viruses in humans and provide important insights for monitoring and evaluating the pandemic potential of field influenza viruses.     

Identification of swine influenza A virus and Stenotrophomonas maltophilia co-infection in Chinese pigs

ABSTRACT: BACKGROUND: Influenza virus virulence can be exacerbated by bacterial co-infections. Swine influenza virus (SIV) infection together with some bacteria is found to enhance pathogenicity. METHODS: SIV-positive samples suspected of containing bacteria were used for bacterial isolation and identification. Antimicrobial susceptibility testing was performed by disc diffusion methods. To investigate the interaction of SIV and the bacteria in vitro, guinea pigs were used as mammalian hosts to determine the effect on viral susceptibility and transmissibility. Differences in viral titers between groups were compared using Student's t-test. RESULTS: During surveillance for SIV in China from 2006 to 2009, seven isolates (24.14%) of 29 influenza A viruses were co-isolated with Stenotrophomonas maltophilia from nasal and tracheal swab samples of pigs. Antimicrobial susceptibility testing showed that the bacteria possessed a high level of resistance towards clinically used antibiotics. To investigate the interaction between these two microorganisms in influencing viral susceptibility and transmission in humans, guinea pigs were used as an infection model. Animals were inoculated with SIV or S. maltophilia alone or co-infected with SIV and S. maltophilia. The results showed that although no transmission among guinea pigs was observed, virus--bacteria co-infections resulted in higher virus titers in nasal washes and trachea and a longer virus shedding period. CONCLUSIONS: This is the first report of influenza virus co-infection with S. maltophilia in the Chinese swine population. Increased replication of virus by co-infection with multidrug resistant bacteria might increase the infection rate of SIV in humans. The control of S. maltophilia in clinics will contribute to reducing the spread of SIV in pigs and humans.     

The ferret as a model organism to study influenza A virus infection

Influenza is a human pathogen that continues to pose a public health threat. The use of small mammalian models has become indispensable for understanding the virulence of influenza viruses. Among numerous species used in the laboratory setting, only the ferret model is equally well suited for studying both the pathogenicity and transmissibility of human and avian influenza viruses. Here, we compare the advantages and limitations of the mouse, ferret and guinea pig models for research with influenza A viruses, emphasizing the multifarious uses of the ferret in the assessment of influenza viruses with pandemic potential. Research performed in the ferret model has provided information, support and guidance for the public health response to influenza viruses in humans. We highlight the recent and emerging uses of this species in influenza virus research that are advancing our understanding of virus-host interactions.     

Characterization of an artificial swine-origin influenza virus with the same gene combination as H1N1/2009 virus: a genesis clue of pandemic strain

Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.     

禽流感与猪流感病毒：跨越物种传播的新认识

今年在墨西哥暴发的流感疫情来源于一种新的流感病毒：甲型H1N1病毒．这种病毒包括人源,禽源和猪源等甲型流感病毒基因片段,为混合毒株．比较了禽、猪和人的流感病毒在其天然宿主中的致病机理,主要目的是评估猪和禽的流感病毒成为人兽共患病的可能性,同时还评估猪在禽流感病毒传入人的过程中可能起到的作用．禽流感和猪流感作为人畜共患疾病,在流感病毒从动物到人的传播过程中起到关键的作用．猪作为流感病毒的中间宿主,具有混合器作用,人、猪、禽流感病毒可在其体内进行基因重排产生新病毒,并可能跨越种间屏障感染人类．但是流感病毒本身的跨越种间障碍传播不足以引起人流感的大暴发,动物流感病毒必须经过显著的遗传变异后才能使其在人群中长期存在．     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they have been proposed to be intermediate hosts or mixing vessels for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.     

Mutations in the NS1 protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs

It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-alpha/beta) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-alpha/beta synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-alpha/beta induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.     

Influenza—an emerging and re-emerging disease

Influenza A viruses continue to emerge from the aquatic avian reservoir and cause pandemics. There are periodic exchanges of influenza virus genes or whole viruses between avians and other species giving rise to pandemics of diseases in humans, lower animals and birds. It is hypothesized that pigs are an intermediate host and that China is an epicenter for the evolution of human pandemic strains. However, the transmission of avian influenza viruses to pigs in Europe in 1979 and detection of reassortants with human influenza genes in pigs raises the question of whether the next pandemic of influenza will emerge in Europe!     

High genetic and antigenic similarity between a swine H3N2 influenza A virus and a prior human influenza vaccine virus: A possible immune pressure-driven cross-species transmission

In late April of 2009, a global outbreak of human influenza was reported. The causative agent is a highly unusual reassortant H1N1 influenza virus carrying genetic segments derived from swine, human and avian influenza viruses. In this study, we compared the HA, NA and other gene segments of a swine H3N2 influenza A virus, A/Swine/Guangdong/z5/2003, which was isolated from pigs in 2003 in Guangdong Province, China, to the predominant human and swine H3N2 viruses. We found that the similarity of gene segments of A/Swine/Guangdong/z5/2003 was closer to Moscow/99-like human H3N2 virus than Europe swine H3N2 viruses during 1999-2002. These results suggest that A/Swine/Guangdong/z5/2003 may be porcine in origin, possibly being driven by human immune pressure induced by either natural H3N2 virus infection or use of A/Moscow/10/99 (H3N2)-based human influenza vaccine. The results further confirm that swine may play a dual role as a “shelter” for hosting influenza virus from humans or birds and as a “mixing vessel” for generating reassortant influenza viruses, such as the one causing current influenza pandemic.     

Influenza A viruses: new research developments

Influenza A viruses are zoonotic pathogens that continuously circulate and change in several animal hosts, including birds, pigs, horses and humans. The emergence of novel virus strains that are capable of causing human epidemics or pandemics is a serious possibility. Here, we discuss the value of surveillance and characterization of naturally occurring influenza viruses, and review the impact that new developments in the laboratory have had on our understanding of the host tropism and virulence of viruses. We also revise the lessons that have been learnt from the pandemic viruses of the past 100 years.     

Reassortant between Human-Like H3N2 and Avian H5 Subtype Influenza A Viruses in Pigs: A Potential Public Health Risk

Background

Human-like H3N2 influenza viruses have repeatedly been transmitted to domestic pigs in different regions of the world, but it is still uncertain whether any of these variants could become established in pig populations. The fact that different subtypes of influenza viruses have been detected in pigs makes them an ideal candidate for the genesis of a possible reassortant virus with both human and avian origins. However, the determination of whether pigs can act as a “mixing vessel” for a possible future pandemic virus is still pending an answer. This prompted us to gather the epidemiological information and investigate the genetic evolution of swine influenza viruses in Jilin, China.

Methods

Nasopharyngeal swabs were collected from pigs with respiratory illness in Jilin province, China from July 2007 to October 2008. All samples were screened for influenza A viruses. Three H3N2 swine influenza virus isolates were analyzed genetically and phylogenetically.

Results

Influenza surveillance of pigs in Jilin province, China revealed that H3N2 influenza viruses were regularly detected from domestic pigs during 2007 to 2008. Phylogenetic analysis revealed that two distinguishable groups of H3N2 influenza viruses were present in pigs: the wholly contemporary human-like H3N2 viruses (represented by the Moscow/10/99-like sublineage) and double-reassortant viruses containing genes from contemporary human H3N2 viruses and avian H5 viruses, both co-circulating in pig populations.

Conclusions

The present study reports for the first time the coexistence of wholly human-like H3N2 viruses and double-reassortant viruses that have emerged in pigs in Jilin, China. It provides updated information on the role of pigs in interspecies transmission and genetic reassortment of influenza viruses.     

Studies of H5N1 influenza virus infection of pigs by using viruses isolated in Vietnam and Thailand in 2004

To determine whether avian H5N1 influenza viruses associated with human infections in Vietnam had transmitted to pigs, we investigated serologic evidence of exposure to H5N1 influenza virus in Vietnamese pigs in 2004. Of the 3,175 pig sera tested, 8 (0.25%) were positive for avian H5N1 influenza viruses isolated in 2004 by virus neutralization assay and Western blot analysis. Experimental studies of replication and transmissibility of the 2004 Asian H5N1 viruses in pigs revealed that all viruses tested replicated in the swine respiratory tract but none were transmitted to contact pigs. Virus titers from nasal swabs peaked on day 2, and low titers were detected in the liver of two of the four pigs tested. Our findings indicate that pigs can be infected with highly lethal Asian H5N1 viruses but that these viruses are not readily transmitted between pigs under experimental conditions.     

A Novel Nonhuman Primate Model for Influenza Transmission

Studies of influenza transmission are necessary to predict the pandemic potential of emerging influenza viruses. Currently, both ferrets and guinea pigs are used in such studies, but these species are distantly related to humans. Nonhuman primates (NHP) share a close phylogenetic relationship with humans and may provide an enhanced means to model the virological and immunological events in influenza virus transmission. Here, for the first time, it was demonstrated that a human influenza virus isolate can productively infect and be transmitted between common marmosets (Callithrix jacchus), a New World monkey species. We inoculated four marmosets with the 2009 pandemic virus A/California/07/2009 (H1N1pdm) and housed each together with a naïve cage mate. We collected bronchoalveolar lavage and nasal wash samples from all animals at regular intervals for three weeks post-inoculation to track virus replication and sequence evolution. The unadapted 2009 H1N1pdm virus replicated to high titers in all four index animals by 1 day post-infection. Infected animals seroconverted and presented human-like symptoms including sneezing, nasal discharge, labored breathing, and lung damage. Transmission occurred in one cohabitating pair. Deep sequencing detected relatively few genetic changes in H1N1pdm viruses replicating in any infected animal. Together our data suggest that human H1N1pdm viruses require little adaptation to replicate and cause disease in marmosets, and that these viruses can be transmitted between animals. Marmosets may therefore be a viable model for studying influenza virus transmission.     

The use of chimeric Venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants

Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.