Search

After an outbreak of pandemic influenza A/H1N1 (pH1N1) virus, we had previously reported the emergence of a recombinant canine influenza virus (CIV) between the pH1N1 virus and the classic H3N2 CIV. Our ongoing routine surveillance isolated another reassortant H3N2 CIV carrying the matrix gene of the pH1N1 virus from 2012. The infection dynamics of this H3N2 CIV variant (CIV/H3N2mv) were investigated in dogs and ferrets via experimental infection and transmission. The CIV/H3N2mv-infected dogs and ferrets produced typical symptoms of respiratory disease, virus shedding, seroconversion, and direct-contact transmissions. Although indirect exposure was not presented for ferrets, CIV/H3N2mv presented higher viral replication in MDCK cells and more efficient transmission was observed in ferrets compared to classic CIV H3N2. This study demonstrates the effect of reassortment of the M gene of pH1N1 in CIV H3N2.

The H7N9 influenza virus caused significant mortality and morbidity in infected humans during an outbreak in China in 2013 stimulating vaccine development efforts. As previous H7-based vaccines have been poorly immunogenic in humans we sought to determine the immunogenic and protective properties of an inactivated whole virus vaccine derived from a 2013 H7N9 virus in ferrets. As whole virus vaccine preparations have been shown to be more immunogenic in humans, but less likely to be used, than split or surface antigen formulations, we vaccinated ferrets with a single dose of 15, 30, or 50 μg of the vaccine and subsequently challenged with wild-type A/Anhui/1/2013 (H7N9) either by direct instillation or by contact with infected animals. Although ferrets vaccinated with higher doses of vaccine had higher serum hemagglutinin inhibition (HI) titers, the titers were still low. During subsequent instillation challenge, however, ferrets vaccinated with 50 μg of vaccine showed no illness and shed significantly less virus than mock vaccinated controls. All vaccinated ferrets had lower virus loads in their lungs as compared to controls. In a separate study where unvaccinated-infected ferrets were placed in the same cage with vaccinated-uninfected ferrets, vaccination did not prevent infection in the contact ferrets, although they showed a trend of lower viral load. Overall, we conclude that inactivated whole-virus H7N9 vaccine was able to reduce the severity of infection and viral load, despite the lack of hemagglutinin-inhibiting antibodies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Recently, novel H7N9 influenza viruses have caused an unprecedented outbreak in humans. Pigs are an important intermediate host for influenza; thus, we assessed the replication ability of three human H7N9 viruses (A/Anhui/1/2013, A/Shanghai/1/2013, A/Shanghai/2/2013) in swine tissue explants. All viruses tested replicated efficiently in explants from tracheas and bronchi, with limited replication in alveolar cells. Swine respiratory tissue explants can serve as an efficient model for screening replication potential of newly emerging H7N9 viruses.

In the past 4 years, incidences of endemic or epidemic respiratory diseases associated with canine influenza H3N2 virus in Asian dogs have been reported in countries such as South Korea and China. Canine species were considered to be the new natural hosts for this virus. However, at the beginning of 2010, influenza-like respiratory signs, such as dyspnoea, were also observed among cats as well as in dogs in an animal shelter located in Seoul, South Korea. The affected cats showed 100 % morbidity and 40 % mortality. We were able to isolate a virus from a lung specimen of a dead cat, which had suffered from the respiratory disease, in embryonated-chicken eggs. The eight viral genes isolated were almost identical to those of the canine influenza H3N2 virus, suggesting interspecies transmission of canine influenza H3N2 virus to the cat. Moreover, three domestic cats infected with intranasal canine/Korea/GCVP01/07 (H3N2) all showed elevated rectal temperatures, nasal virus shedding and severe pulmonary lesions, such as suppurative bronchopneumonia. Our study shows, for the first time, that cats are susceptible to canine influenza H3N2 infection, suggesting that cats may play an intermediate host role in transmitting the H3N2 virus among feline and canine species, which could lead to the endemic establishment of the virus in companion animals. Such a scenario raises a public health concern, as the possibility of the emergence of new recombinant feline or canine influenza viruses in companion animals with the potential to act as a zoonotic infection cannot be excluded.

Unlabelled: The genotype of the host is one of several factors involved in the pathogenesis of an infectious disease and may be a key parameter in the epidemiology of highly pathogenic H5N1 influenza virus infection in humans. Gene polymorphisms may affect the viral replication rate or alter the host's immune response to the virus. In humans, it is unclear which aspect dictates the severity of H5N1 virus disease. To identify the mechanism underlying differential responses to H5N1 virus infection in a genetically diverse population, we assessed the host responses and lung viral loads in 21 inbred mouse strains upon intranasal inoculation with A/Hong Kong/213/03 (H5N1). Resistant mouse strains survived large inocula while susceptible strains succumbed to infection with 1,000- to 10,000-fold-lower doses. Quantitative analysis of the viral load after inoculation with an intermediate dose found significant associations with lethality as early as 2 days postinoculation, earlier than any other disease indicator. The increased viral titers in the highly susceptible strains mediated a hyperinflamed environment, indicated by the distinct expression profiles and increased production of inflammatory mediators on day 3. Supporting the hypothesis that viral load rather than an inappropriate response to the virus was the key severity-determining factor, we performed quantitative real-time PCR measuring the cytokine/viral RNA ratio. No significant differences between susceptible and resistant mouse strains were detected, confirming that it is the host genetic component controlling viral load, and therefore replication dynamics, that is primarily responsible for a host's susceptibility to a given H5N1 virus., Importance: Highly pathogenic H5N1 influenza virus has circulated in Southeast Asia since 2003 but has been confirmed in relatively few individuals. It has been postulated that host genetic polymorphisms increase the susceptibility to infection and severe disease. The mechanisms and host proteins affected during severe disease are unknown. Inbred mouse strains vary considerably in their ability to resist H5N1 virus and were used to identify the primary mechanism determining disease severity. After inoculation with H5N1, resistant mouse strains had reduced amounts of virus in their lungs, which subsequently resulted in lower production of proinflammatory mediators and less pathology. We therefore conclude that the host genetic component controlling disease severity is primarily influencing viral replication. This is an important concept, as it emphasizes the need to limit virus replication through antiviral therapies and it shows that the hyperinflammatory environment is simply a reflection of more viral genetic material inducing a response.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

One of the greatest influenza pandemic threats at this time is posed by the highly pathogenic H5N1 avian influenza viruses. To date, 61% of the 433 known human cases of H5N1 infection have proved fatal. Animals infected by H5N1 viruses have demonstrated acute neurological signs ranging from mild encephalitis to motor disturbances to coma. However, no studies have examined the longer-term neurologic consequences of H5N1 infection among surviving hosts. Using the C57BL/6J mouse, a mouse strain that can be infected by the A/Vietnam/1203/04 H5N1 virus without adaptation, we show that this virus travels from the peripheral nervous system into the CNS to higher levels of the neuroaxis. In regions infected by H5N1 virus, we observe activation of microglia and alpha-synuclein phosphorylation and aggregation that persists long after resolution of the infection. We also observe a significant loss of dopaminergic neurons in the substantia nigra pars compacta 60 days after infection. Our results suggest that a pandemic H5N1 pathogen, or other neurotropic influenza virus, could initiate CNS disorders of protein aggregation including Parkinson's and Alzheimer's diseases.

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.

Influenza viruses belong to the family Orthomyxoviridae. Genus Influenza A viruses are true zoonotic agents with many animal reservoirs, whereas genus Influenza B viruses are generally considered to be a virus of humans. The genome of influenza A viruses consists of eight unique segments of single-stranded RNA of negative polarity; they are typed according to their surface proteins, hemagglutinin (HA) and neuraminidase (NA). HA and NA, the major antigenic determinants of influenza A viruses, are present in 16 and nine serologic subtypes, respectively. Annual epidemics and occasional pandemics of influenza in humans depend on the continued evolution of influenza viruses. Although they have numerous potential host populations, most of our genetic and biologic data are obtained from studies of domestic populations of species such as chickens, turkeys, swine, and horses. Concerning wildlife populations, including wild populations of these domesticated species, much less is known. The purpose of this review is to establish what role wildlife populations play in the continued evolution of influenza viruses. Future work needs to determine what chain of events makes it possible for an influenza virus to be successfully transmitted to, and more importantly within, an alternative host population. Even questions as fundamental as which hosts can transmit viruses to humans remain unanswered so far.

Background: In response to the emergence of severe infection capable of rapid global spread, WHO will issue a pandemic alert. Such alerts are rare; however, on Feb 19, 2003, a pandemic alert was issued in response to human infections caused by an avian H5N1 influenza virus, A/Hong Kong/213/03. H5N1 had been noted once before in human beings in 1997 and killed a third (6/18) of infected people. The 2003 variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. Candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. Since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics., Methods: We removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of A/Hong Kong/213/03 using influenza reverse genetics techniques. A reference vaccine virus was then produced on an A/Puerto Rico/8/34 (PR8) backbone on WHO-approved Vero cells. We assessed this reference virus for pathogenicity in in-vivo and in-vitro assays., Findings: A reference vaccine virus was produced in Good Manufacturing Practice (GMP)-grade facilities in less than 4 weeks from the time of virus isolation. This virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs., Interpretation: The ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. The same technologies and procedures are currently being used to create reference vaccine viruses against the 2004 H5N1 viruses circulating in Asia.

The orally administered neuraminidase (NA) inhibitor RWJ-270201 was tested in parallel with zanamivir and oseltamivir against a panel of avian influenza viruses for inhibition of NA activity and replication in tissue culture. The agents were then tested for protection of mice against lethal H5N1 and H9N2 virus infection. In vitro, RWJ-270201 was highly effective against all nine NA subtypes. NA inhibition by RWJ-270201 (50% inhibitory concentration, 0.9 to 4.3 nM) was superior to that by zanamivir and oseltamivir carboxylate. RWJ-270201 inhibited the replication of avian influenza viruses of both Eurasian and American lineages in MDCK cells (50% effective concentration, 0.5 to 11.8 microM). Mice given 10 mg of RWJ-270201 per kg of body weight per day were completely protected against lethal challenge with influenza A/Hong Kong/156/97 (H5N1) and A/quail/Hong Kong/G1/97 (H9N2) viruses. Both RWJ-270201 and oseltamivir significantly reduced virus titers in mouse lungs at daily dosages of 1.0 and 10 mg/kg and prevented the spread of virus to the brain. When treatment began 48 h after exposure to H5N1 virus, 10 mg of RWJ-270201/kg/day protected 50% of mice from death. These results suggest that RWJ-270201 is at least as effective as either zanamivir or oseltamivir against avian influenza viruses and may be of potential clinical use for treatment of emerging influenza viruses that may be transmitted from birds to humans.

We established a porcine lung epithelial cell line designated St. Jude porcine lung cells (SJPL) and demonstrated that all tested influenza A and B viruses replicated in this cell line. The infectivity titers of most viruses in SJPL cells were comparable to or better than those in MDCK cells. The propagation of influenza viruses from clinical samples in SJPL cells did not lead to antigenic changes in the hemagglutinin molecule. The numbers of both Sia2-3Gal and Sia2-6Gal receptors on SJPL cells were greater than those on MDCK cells. Influenza virus infection of SJPL cells did not lead to apoptosis, as did infection of MDCK cells. No porcine endogenous retrovirus was detected in SJPL cells, and in contrast to MDCK cells, SJPL cells did not cause tumors in nude mice.

In 1997, an avian H5N1 influenza virus, A/Hong Kong/156/97 (A/HK/156/97), caused six deaths in Hong Kong, and in 1999, an avian H9N2 influenza virus infected two children in Hong Kong. These viruses and a third avian virus [A/Teal/HK/W312/97 (H6N1)] have six highly related genes encoding internal proteins. Additionally, A/Chicken/HK/G9/97 (H9N2) virus has PB1 and PB2 genes that are highly related to those of A/HK/156/97 (H5N1), A/Teal/HK/W312/97 (H6N1), and A/Quail/HK/G1/97 (H9N2) viruses. Because of their similarities with the H5N1 virus, these H6N1 and H9N2 viruses may have the potential for interspecies transmission. We demonstrate that these H6N1 and H9N2 viruses are pathogenic in mice but that their pathogenicities are less than that of A/HK/156/97 (H5N1). Unadapted virus replicated in lungs, but only A/HK/156/97 (H5N1) was found in the brain. After three passages (P3) in mouse lungs, the pathogenicity of the viruses increased, with both A/Teal/HK/W312/97 (H6N1) (P3) and A/Quail/HK/G1/97 (H9N2) (P3) viruses being found in the brain. The neuraminidase inhibitor zanamivir inhibited viral replication in Madin-Darby canine kidney cells in virus yield assays (50% effective concentration, 8.5 to 14.0 microM) and inhibited viral neuraminidase activity (50% inhibitory concentration, 5 to 10 nM). Twice daily intranasal administration of zanamivir (50 and 100 mg/kg of body weight) completely protected infected mice from death. At a dose of 10 mg/kg, zanamivir completely protected mice from infection with H9N2 viruses and increased the mean survival day and the number of survivors infected with H6N1 and H5N1 viruses. Zanamivir, at all doses tested, significantly reduced the virus titers in the lungs and completely blocked the spread of virus to the brain. Thus, zanamivir is efficacious in treating avian influenza viruses that can be transmitted to mammals.

During 1998, severe outbreaks of influenza were observed in four swine herds in the United States. This event was unique because the causative agents, H3N2 influenza viruses, are infrequently isolated from swine in North America. Two antigenically distinct reassortant viruses (H3N2) were isolated from infected animals: a double-reassortant virus containing genes similar to those of human and swine viruses, and a triple-reassortant virus containing genes similar to those of human, swine, and avian influenza viruses (N. N. Zhou, D. A. Senne, J. S. Landgraf, S. L. Swenson, G. Erickson, K. Rossow, L. Liu, K.-J. Yoon, S. Krauss, and R. G. Webster, J. Virol. 73:8851-8856, 1999). Because the U.S. pig population was essentially naive in regard to H3N2 viruses, it was important to determine the extent of viral spread. Hemagglutination inhibition (HI) assays of 4, 382 serum samples from swine in 23 states indicated that 28.3% of these animals had been exposed to classical swine-like H1N1 viruses and 20.5% had been exposed to the triple-reassortant-like H3N2 viruses. The HI data suggested that viruses antigenically related to the double-reassortant H3N2 virus have not become widespread in the U.S. swine population. The seroreactivity levels in swine serum samples and the nucleotide sequences of six additional 1999 isolates, all of which were of the triple-reassortant genotype, suggested that H3N2 viruses containing avian PA and PB2 genes had spread throughout much of the country. These avian-like genes cluster with genes from North American avian viruses. The worldwide predominance of swine viruses containing an avian-like internal gene component suggests that these genes may confer a selective advantage in pigs. Analysis of the 1999 swine H3N2 isolates showed that the internal gene complex of the triple-reassortant viruses was associated with three recent phylogenetically distinct human-like hemagglutinin (HA) molecules. Acquisition of HA genes from the human virus reservoir will significantly affect the efficacy of the current swine H3N2 vaccines. This finding supports continued surveillance of U.S. swine populations for influenza virus activity.

In order to assess the degree of immune cross-protection among avian H2 influenza virus strains, mice were immunised with beta-propiolactone-inactivated virus preparations and infected intranasally with mouse-adapted variant of A/Black Duck/New Jersey/1580/78 (H2N3) strain. The experiments with 11 avian H2 strains revealed that both Eurasian and American H2 avian influenza viruses exhibit either high or moderate degree of cross-protection. The grouping of the strains in accordance with their cross-protection efficiency does not coincide with H2 phylogenetic branches. Several reassortant clones were obtained with the use of A/Pintail Duck/Primorie/695/76 (H2N3) strain and high-yield X-67 reassortant as parent viruses, among them a high-yield H2N3 reassortant. Taking into account the data on cross-protection among avian H2 strains, the high-yield H2N3 reassortant may be regarded as a prototype strain to be used for the preparation of killed vaccines in the case of a new appearance of avian H2 haemagglutinin in circulation in humans.

In Hong Kong in 1997, a highly lethal H5N1 avian influenza virus was apparently transmitted directly from chickens to humans with no intermediate mammalian host and caused 18 confirmed infections and six deaths. Strategies must be developed to deal with this virus if it should reappear, and prospective vaccines must be developed to anticipate a future pandemic. We have determined that unadapted H5N1 viruses are pathogenic in mice, which provides a well-defined mammalian system for immunological studies of lethal avian influenza virus infection. We report that a DNA vaccine encoding hemagglutinin from the index human influenza isolate A/HK/156/97 provides immunity against H5N1 infection of mice. This immunity was induced against both the homologous A/HK/156/97 (H5N1) virus, which has no glycosylation site at residue 154, and chicken isolate A/Ck/HK/258/97 (H5N1), which does have a glycosylation site at residue 154. The mouse model system should allow rapid evaluation of the vaccine's protective efficacy in a mammalian host. In our previous study using an avian model, DNA encoding hemagglutinin conferred protection against challenge with antigenic variants that differed from the primary antigen by 11 to 13% in the HA1 region. However, in our current study we found that a DNA vaccine encoding the hemagglutinin from A/Ty/Ir/1/83 (H5N8), which differs from A/HK/156/97 (H5N1) by 12% in HA1, prevented death but not H5N1 infection in mice. Therefore, a DNA vaccine made with a heterologous H5 strain did not prevent infection by H5N1 avian influenza viruses in mice but was useful in preventing death.

In 1979, an H1N1 avian influenza virus crossed the species barrier, establishing a new lineage in European swine. Because there is no direct or serologic evidence of previous H1N1 strains in these pigs, these isolates provide a model for studying early evolution of influenza viruses. The evolutionary rates of both the coding and noncoding changes of the H1N1 swine strains are higher than those of human and classic swine influenza A viruses. In addition, early H1N1 swine isolates show a marked plaque heterogeneity that consistently appears after a few passages. The presence of a mutator mutation was postulated (C. Scholtissek, S. Ludwig, and W. M. Fitch, Arch. Virol. 131:237-250, 1993) to account for these observations and the successful establishment of an avian H1N1 strain in swine. To address this question, we calculated the mutation rates of A/Mallard/New York/6750/78 (H2N2) and A/Swine/Germany/2/81 (H1N1) by using the frequency of amantadine-resistant mutants. To account for the inherent variability of estimated mutation rates, we used a probabilistic model for the statistical analysis. The resulting estimated mutation rates of the two strains were not significantly different. Therefore, an increased mutation rate due to the presence of a mutator mutation is unlikely to have led to the successful introduction of avian H1N1 viruses in European swine.

Zanamivir, a neuraminidase inhibitor, has shown promise as a drug to control influenza. During prolonged treatment with zanamivir, a mutant virus was isolated from an immunocompromised child infected with influenza B virus. A hemagglutinin mutation (198 Thr-->Ile) reduced the virus affinity for receptors found on susceptible human cells. A mutation in the neuraminidase active site (152 Arg-->Lys) led to a 1000-fold reduction in the enzyme sensitivity to zanamivir. When tested in ferrets, the mutant virus had less virulence than the parent; however, it had a growth preference over the parent in zanamivir-treated animals. Despite these changes, the sensitivity of the mutant virus to zanamivir assessed by a standard test in MDCK cells was unaffected. These data indicate that the current methods for monitoring resistant mutants are potentially flawed because no tissue culture system adequately reflects the receptor specificity of human respiratory tract epithelium.

Coinfection of wild birds by influenza A viruses is thought to be an important mechanism for the diversification of viral phenotypes by generation of reassortants. However, it is not known whether coinfection is a random event or follows discernible patterns with biological significance. In the present study, conducted with viruses collected throughout 15 years from a wild-duck population in Alberta, Canada, we identified three discrete distributions of coinfections. In about one-third of the events, which involved subtypes of viruses that appear to be maintained in this duck reservoir, coinfection occurred at rates either close to or significantly lower than one would predict from rates of single-virus infection. Apparently, the better adapted an influenza A virus is to an avian population, the greater is its ability to prevent coinfections. Conversely, poorly adapted, nonmaintained viruses were significantly overrepresented as coinfectants. Rarely encountered subtypes appear to represent viruses whose chances of successfully infiltrating avian reservoirs are increased by coinfection. Mallards (Anas platyrhynchos) and pintails (A. acuta) were significantly more likely to be infected by a single influenza A virus than were the other species sampled, but no species was significantly more likely to be coinfected. These observations provide the first evidence of nonrandom coinfection of wild birds by influenza A viruses, suggesting that reassortment of these viruses in a natural population does not occur randomly. These results suggest that even though infections may occur in a species, all subtypes are not maintained by all avian species. They also suggest that specific influenza A virus subtypes are differentially adapted to different avian hosts and that the fact that a particular subtype is isolated from a particular avian species does not mean that the virus is maintained by that species.

In May 1993, a severe epidemic of respiratory disease began in horses in Inner Mongolia and spread throughout horses in China. The disease affected mules and donkeys as well as horses but did not spread to other species, including humans. The severity of the disease raised the question of whether the outbreak might have been caused by the new avian-like influenza viruses detected in horses in China in 1989 or by current variants ofA/equine/Miami/1/63 (H3N8) (equine-2) or by a reassortant between these viruses. Antigenic and sequence analysis established that all gene segments of the influenza virus causing the epidemic were of recent equine-2 origin and that the virus was not a reassortant. Serological analysis of post-infection horse sera provided evidence for the continued circulation of the A/Equine/Jilin/1/89 (Eq/Jilin) (H3N8) avian-like viruses in horses in Heilongjiang province with original antigenic sin-like responses. It is noteworthy that prior infection with the avian-like Eq/Jilin strain did not afford cross-protection against a current equine-2 strain. Serological evidence for the continued circulation of the avian-like H3N8 influenza virus in horses indicates that this virus has probably established itself in horses in Asia.

To characterize behavioral and physiological alterations induced by viral respiratory infection, C57BL/6 and BALB/c strains of mice were monitored for 2 days before and 4 days after intranasal inoculation with influenza virus. Both strains developed hypothermia, decrease locomotor activity, and decreased delta-wave amplitude during sleep within 24 h after inoculation. However, infected C57BL/6 mice also spent more time in slow-wave sleep, but infected BALB/c mice did not. The increased SWS in C57BL/6 mice occurred during the lights-off phase of the circadian cycle, and resulted in loss of the normal circadian rhythmicity of sleep. Increased sleep also occurred after viral challenge of immunized C57BL/6 mice, but was not observed after secondary challenge of immunized BALB/c mice. These data indicate that sleep alterations may accompany viral infections in some, but not all, strains of mice. The dissimilar sleep patterns seen in C57BL/6 and BALB/c mice after influenza infection may reflect differences in their immune response to influenza virus.

Direct DNA inoculations are being developed as a method of subunit vaccination. Plasmid DNAs encoding influenza virus hemagglutinin glycoproteins have been tested for the ability to provide protection against lethal influenza challenges. In immunization trials using inoculations of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens were protected against the lethal challenge. Good protection was achieved by intramuscular, intravenous and intradermal injections. In mice, 95% protection was achieved by gene gun delivery of 250-2500 times less DNA than the saline inoculations. Successful DNA vaccination by multiple routes of inoculation and the high efficiency of gene-gun delivery highlight the potential of this promising new approach to immunization.

The Asian/57, Hong Kong/68, and Russian/77 pandemics of this century appeared or reappeared in China. Interspecies transmission and genetic reassortment of influenza viruses have been implicated in the origin of these human pandemic influenza viruses. Pigs have been suspected to be the "mixing vessel" where reassortment occurs. To investigate this possibility, 104 porcine influenza viruses collected at random from Southern China from 1976 to 1982, including 32 H3N2 isolates and 72 H1N1 isolates, were studied using dot blot hybridization, partial sequencing, and phylogenetic analysis. There were 29 of 32 H3N2 isolates characteristic of viruses originally derived from humans; the other 3 isolates were reassortants containing genes from porcine and human influenza viruses. Phylogenetic analyses of the polymerase B1 (PB1) genes showed that interspecies transmission from humans to pigs has happened multiple times in pigs in Southern China. All 72 H1N1 isolates were of porcine origin characteristic of classical porcine H1N1 influenza virus. Analysis of 624 genes of porcine influenza viruses from Southern China failed to detect any evidence for avian influenza virus genes. This contrasts to what is currently found in Europe, where the majority of porcine influenza virus isolates are of avian origin.

Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 micrograms of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 micrograms of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines.

Growth of clinical specimens of influenza viruses in eggs can result in the selection of antigenic variants distinct from corresponding viruses grown in mammalian tissue culture. To evaluate the contribution of host cell selection on the antigenic diversity of human influenza isolates, as seen in annual surveillance studies, viruses grown in embryonated eggs were compared by antigenic and genetic analyses with their mammalian tissue culture-grown counterparts. Clinical specimens were gathered from around the world from late 1987 to 1990 and the antigenicity of isolated viruses was assessed by hemagglutination-inhibition assays using immune ferret sera as is currently performed for routine surveillance and the selection of vaccine strains. In addition, viruses were assessed using a panel of anti-H3 HA monoclonal antibodies. The extent of antigenic variation exhibited by the egg-grown strains was far greater than the relative antigenic homogeneity of the tissue culture-grown viruses. Nucleotide sequence analysis of HA1 gene PCR products of 28 MDCK cell and egg derived pairs allowed identification of amino acid substitutions responsible for the antigenic differences observed and the adaptation to growth in eggs. Among these substitutions was a change at amino acid position 186 of HA1 (Ser in tissue culture viruses and lle in egg-grown viruses) which was observed at relatively high frequency. Egg- and MDCK-grown pairs with this single amino acid difference were classified into distinct antigenic groups by ferret sera raised to WHO reference viruses. Given the additional antigenic diversity observed among egg-grown strains, considerable care should be taken in the selection of reference and vaccine strains grown in eggs. Rapid sequence comparisons of MDCK- and egg-grown viruses allow identification of variants arising through egg selection and will prove to be a useful adjunct to antigenic surveillance for the selection of reference and vaccine strains.

H2N2 influenza A viruses caused the Asian pandemic of 1957 and then disappeared from the human population 10 years later. To assess the potential for similar outbreaks in the future, we determined the antigenicity of H2 hemagglutinins (HAs) from representative human and avian H2 viruses and then analyzed the nucleotide and amino acid sequences to determine their evolutionary characteristics in different hosts. The results of longitudinal virus surveillance studies were also examined to estimate the prevalence of avian H2 isolates among samples collected from wild ducks and domestic poultry. Reactivity patterns obtained with a large panel of monoclonal antibodies indicated antigenic drift in the HA of human H2 influenza viruses, beginning in 1962. Amino acid changes were clustered in two regions of HA1 that correspond to antigenic sites A and D of the H3 HA. By contrast, the antigenic profiles of the majority of avian H2 HAs were remarkably conserved through 1991, resembling the prototype Japan 57 (H2N2) strain. Amino acid changes were distributed throughout HA1, indicating that antibodies do not play a major role in the selection of avian H2 viruses. Phylogenetic analysis revealed two geographic site-specific lineages of avian H2 HAs: North American and Eurasian. Evidence is presented to support interregion transmission of gull H2 viruses. The human H2 HAs that circulated in 1957-1968 form a separate phylogenetic lineage, most closely related to the Eurasian avian H2 HAs. There was an increased prevalence of H2 influenza viruses among wild ducks in 1988 in North America, preceding the appearance of H2N2 viruses in domestic fowl. As the prevalence of avian H2N2 influenza viruses increased on turkey farms and in live bird markets in New York City and elsewhere, greater numbers of these viruses have come into direct contact with susceptible humans. We conclude that antigenically conserved counterparts of the human Asian pandemic strain of 1957 continue to circulate in the avian reservoir and are coming into closer proximity to susceptible human populations.

Direct DNA inoculations have been used to demonstrate that in vivo transfections can be used to elicit protective immune responses. The direct inoculation of an H7 haemagglutinin-expressing DNA protected chickens against lethal challenge with an H7N7 influenza virus. Three-week-old chickens were vaccinated by inoculating 100 micrograms of plasma DNA by each of three routes (intravenous, intraperitoneal and subcutaneous). One month later, chickens were boosted with 100 micrograms of DNA by each of the three routes. At 1-2 weeks postboost, chickens were challenged via the nares with 100 lethal doses of an H7N7 virus. Low to undetectable levels of H7-specific antibodies were present postvaccination and boost. High titres of H7-specific antibodies appeared within 1 week of challenge. In a series of four experiments, 50% (28/56) of the DNA-vaccinated and < 2% (1/67) of the control chickens survived the challenge. This exceptionally simple method of immunization holds high promise for the development of subunit vaccines.

An influenza virus, A/equine/Alaska/1/91 (H3N8), was isolated from horses from Alaska with an acute respiratory infection. Pathogenic and serologic studies revealed that this virus is similar to previously isolated equine H3N8 influenza viruses. Antigenic analyses utilizing hemagglutination inhibition and neuraminidase inhibition assays indicated an antigenic drift from the prototype equine H3N8 influenza virus, A/equine/Miami/1/63. Partial sequence analysis of the A/equine/Alaska influenza virus indicated that each of 8 gene sequences are of equine origin.

A new H3N8 equine influenza virus [A/Equine/Jilin/1/89 (Eq/Jilin)] appeared in Northeastern China in 1989 and caused high mortality in horses; the available evidence indicates that it has not yet spread outside this region of the world. Serological analysis with postinfection ferret sera in haemagglutination inhibition (HI) tests confirmed that Eq/Jilin is antigenically distinct from H3N8 equine influenza viruses isolated between 1963 and 1991 and also showed that a current equine influenza virus [A/Equine/Alaska/1/91 (H3N8)] had undergone antigenic drift. In the present study we determine if vaccine against a recent H3N8 influenza virus [A/Equine/Kentucky/1277/90 (Eq/Kentucky)] that was standardized for haemagglutinin content will protect mice against lethal challenge with the new H3N8 influenza virus from China. Complete protection is defined as prevention of virus replication in the lungs of mice 3 days after challenge. High doses of Eq/Kentucky vaccine in aqueous suspension (0.5-5.0 micrograms HA per dose) provided minimal protection against Eq/Jilin challenge as judged by virus titres in the lungs of vaccinated animals. Eq/Kentucky vaccine in adjuvant (1.0-5.0 micrograms HA per dose) did provide complete protection against challenge with Eq/Jilin in mice. Eq/Jilin vaccine in aqueous suspension induced complete protection of mice against challenge with Eq/Kentucky at doses from 0.5 to 5 micrograms HA and in adjuvant doses of Eq/Jilin from 0.1-5.0 micrograms HA were efficacious. Homologous protection against Eq/Jilin or Eq/Kentucky was induced by doses of vaccine from 0.5-5.0 micrograms HA per dose in aqueous suspension and from 0.01-5.0 micrograms HA per dose in adjuvant.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic reassortment between influenza A viruses in humans and in animals and birds has been implicated in the appearance of new pandemics of human influenza. To determine whether such reassortment has occurred in the United States, the authors compared the genetic origins of gene segments of 73 swine influenza virus isolates (1976-1990), representing 11 states, and 11 turkey virus isolates (1980-1989), representing eight states. The host origin of gene segments encoding the internal proteins of H1N1 swine and turkey influenza viruses was identified by developing a dot-blot assay. All gene segments of swine influenza viruses were characteristic of influenza virus genes from that species, indicating that pigs may not be frequent participants in interspecies genetic exchange and reassortment of influenza viruses in the United States. In contrast, 73% of the turkey influenza virus isolates contained genes of swine origin. One turkey isolate was a reassortant having three genes characteristic of avian influenza virus and three of swine origin. These findings document a high degree of genetic exchange and reassortment of influenza A viruses in domestic turkeys in the United States. The molecular biologic techniques used by the authors should aid future epidemiologic studies of influenza pandemics.

In March 1989 a severe outbreak of respiratory disease occurred in horses in the Jilin and Heilongjiang provinces of Northeast China that caused up to 20% mortality in some herds. An influenza virus of the H3N8 subtype was isolated from the infected animals and was antigenically and molecularly distinguishable from the equine 2 (H3N8) viruses currently circulating in the world. The reference strain A/Equine/Jilin/1/89 (H3N8) was most closely related to avian H3N8 influenza viruses. Sequence comparisons of the entire hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and NS genes along with partial sequences of the three polymerase (PB1, PB2, PA) genes suggest that six of the eight gene segments (PA, HA, NP, NA, M, NS) are closely related to avian influenza viruses. Since direct sequence analysis can only provide a crude measure of relationship, phylogenetic analysis was done on the sequence information. Phylogenetic analyses of the entire HA, NP, M, and NS genes and of partial sequences of PB1, PB2, and PA indicated that these genes are of recent avian origin. The NP gene segment is closely related to the gene segment found in the newly described H14 subtype isolated from ducks in the USSR. The A/Equine/Jilin/1/89 (H3N8) influenza virus failed to replicate in ducks, but did replicate and cause disease in mice on initial inoculation and on subsequent passaging caused 100% mortality. In ferrets, the virus caused severe influenza symptoms. A second outbreak of influenza in horses in Northeast China occurred in April 1990 in the Heilongjiang province with 48% morbidity and no mortality. The viruses isolated from this outbreak were antigenically indistinguishable from those in the 1989 outbreak and it is probable that the reduced mortality was due to the immune status of of the horses in the region. No influenza was detected in horses in Northern China in the spring, summer, or fall of 1991 and no influenza has been detected in horses in adjacent areas. Our analysis suggests that this new equine influenza virus in horses in Northeast China is the latest influenza virus in mammals to emerge from the avian gene pool in nature and that it may have spread to horses without reassortment. The appearance of this new equine virus in China emphasizes the potential for whole avian influenza viruses to successfully enter mammalian hosts and serves as a model and a warning for the appearance of new pandemic influenza viruses in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

The influenza A/chicken/Pennsylvania/1/83 (H5N2) virus is the first known example of an influenza virus isolated from a natural infection which contained primarily defective interfering particles (T. M. Chambers and R. G. Webster, J. Virol. 61, 1517-1523, 1987). In chickens, coinoculation of this virus together with the closely related but highly virulent influenza A/chicken/Pennsylvania/1370/83 virus results in reduced mortality compared to virulent virus infection alone (Bean et al., J. Virol. 54, 151-160, 1985). The biological basis of this protective effect has not been established. Protective activity required greater than or equal to 100-fold excess input of protecting virus over virulent virus, functioned effectively during the first generations of virulent virus multiplication, and also functioned against an antigenically heterologous (H7N7) virulent influenza virus. Protection was correlated with the complete inhibition of virulent virus spread to the brain of infected chickens. Plaque-purified chicken/Pennsylvania/1/83 virus depleted of defective interfering particles, and beta-propiolactone-inactivated virus, had no protective effect. These characteristics are consistent with the hypothesis that protection was the result of defective interfering particle-mediated interference with virulent virus multiplication within the respiratory tract of the chicken.

The distribution and clearance of viral RNA (vRNA) and mRNA has been analysed for the acute and recovery stages of the pneumonia induced by intranasal infection of C57BL/6J mice with H3N2 influenza A viruses. Amplification of viral genomic material by the polymerase chain reaction showed that the influenza haemagglutinin (HA) gene was eliminated from the lungs of immunologically intact mice by day 14 post-infection, whereas in vitro depletion of the CD4+ T cells delayed clearance by at most 4 days. Viral RNA encoding the HA gene was first demonstrated in the regional mediastinal lymph nodes at 48 h, and continued to be present until day 6 or day 10 after infection of the intact and CD4-depleted mice, respectively. Evidence for the presence of vRNA in the thymus, but not in the mesenteric lymph nodes or the spleen, was found in some situations. Otherwise, the distribution and clearance of vRNA was as would be predicted from earlier studies using virus isolation procedures to monitor localization patterns, and shows a lack of long-term persistence of the influenza virus genome.

The hemagglutinin (HA) and neuraminidase (NA) of influenza A viruses induce antibodies which augment the uptake of influenza A virus by antigen presenting cells via Fc receptor entry. Antibody-dependent enhancement of uptake of virus by cells was mediated by Fc receptors because F(ab')2 preparations of lgG mixed with virus did not enhance virus uptake. The enhanced infection was measured using a fluorescent focus assay and was confirmed by dot-blot hybridization analysis. A 25-fold increase in the number of cells containing influenza antigens was detected when virus was mixed with subneutralizing concentrations of immune serum to the homologous virus before adding to neuraminidase-treated cells. Infection was also augmented using reassortant viruses which shared only the HA or the NA of the virus used to induce antibodies. Specific antisera to purified HA or NA also enhanced virus uptake. These results indicate that both the HA and the NA induce antibodies that enhance uptake of virus by Fc receptor bearing cells. In addition we determined that the drift of neutralizing antigens occurred more quickly than the drift of infection-enhancing antigens during the evolution of virus strains of the H3 subtype. The increase in the number of antigen presenting cells as a result of uptake of virus complexed with cross-reactive enhancing antibodies may affect the T cell responses to influenza infection.

Swine are an animal reservoir for influenza viruses capable of causing disease in humans. A serological survey in 1988-1989 demonstrates that subtype H1 influenza viruses continue to circulate at high frequency among swine in the north-central U.S.A. (average 51% incidence). Subtype H3 viruses antigenically similar to current human H3 viruses are circulating at low frequency (average 1.1%), particularly in the southeast U.S.A.

The nucleoprotein (NP) genes of influenza viruses were sequenced from a variety of virus isolates derived from marine mammals: whales from the Pacific and Atlantic oceans, seal and gull from the Western Atlantic, and a tern from the Caspian Sea. In comparison to published NP sequences, we found pairs of NPs derived from avian and marine mammal isolates to be closely related, e.g., the gull-whale and mallard-seal pairs from the Atlantic Coast of the USA and the tern-Pacific Ocean whale pair of the Eastern Hemisphere. Our analysis suggests that influenza viruses have been independently introduced into marine mammals from avian sources for each of our three examples. Furthermore, the closeness of the relationship in these avian-mammalian NP pairs indicates that the introductions are relatively recent. The sequences of these marine mammal NPs are avian-like and can be clearly distinguished from human NPs. Our results provide further support of interspecies transmission of influenza A viruses from the avian host reservoir directly to mammalian hosts.

More than 400 harbor seals, most of them immature, died along the New England coast between December 1979 and October 1980 of acute pneumonia associated with influenza virus, A/Seal/Mass/1/180 (H7N7). The virus has avian characteristics, replicates principally in mammals, and causes mild respiratory disease in experimentally infected seals. Concurrent infection with a previously undescribed mycoplasma or adverse environmental conditions may have triggered the epizootic. The similarities between this epizootic and other seal mortalities in the past suggest that these events may be linked by common biological and environmental factors.

Avian influenza viruses replicate in a variety of mammals and birds, yet hemagglutination inhibition tests show that postinfection sera from these animals (e.g., ferrets and ducks) have insignificant levels of antibodies (Hinshaw et al., Infect. Immun. 34:354-361, 1981). This suggested that avian influenza viruses, in contrast to mammalian viruses, may not induce a significant humoral response. Studies reported here indicate that avian influenza viruses do induce high levels of antibodies in ferrets, ducks, and mice and produce long-lived memory for cytotoxic T-cells in mice. The failure to detect hemagglutination-inhibiting antibodies to avian viruses was explained by the finding that antibodies to avian influenza viruses were not detectable in hemagglutination inhibition tests with intact virus yet were readily demonstrable when hemagglutinin subunits were used. In addition, these sera contained high levels of neutralizing antibodies to the avian virus. These findings suggest that the hemagglutinins of avian and mammalian influenza viruses may differ in their accessibility to antibodies or the biological consequence of antibody attachment or both. The practical consequence of these studies is that hemagglutination inhibition tests with intact avian viruses fail to detect antibody and do not correlate with virus neutralization. The avian virus used in these studies, A/Mallard/NY/6870/78 (H2N2), replicated and caused mortality in BALB/c mice, emphasizing that the host range and virulence of avian viruses extends to mammals. The above findings suggest that avian viruses could infect mammals in nature, yet seroepidemiological studies with conventional hemagglutination inhibition tests could give misleading results.

A severe influenza outbreak occurred in horses in South Africa in 1986. The causative agent was identified as an influenza virus [A/Equine/Johannesburg/86 (H3N8)]. Antigenic analyses of the hemagglutinin (HA) with ferret antisera and monoclonal antibodies showed that the Eq/Johannesburg/86 virus is similar to recent equine H3 viruses. The nucleotide sequence analysis on the HA genes of Eq/Johannesburg/86 and other equine H3 influenza viruses, together with the epidemiological data, clearly demonstrated that the Eq/Johannesburg/86 virus was derived from a virus that had been circulating in horses in the United States in 1986-87. The epidemiological information suggests that the unusually severe influenza outbreak in South Africa may be due to the lack of immunity to these viruses in the horse population.

Fowlpox virus, the prototypic virus of the genus Avipoxvirus has a natural host range limited to avian species. As such, fowlpox virus provides a suitable candidate for the development of a species-specific recombinant viral vector. This paper reports the development of a fowlpox virus recombinant expressing the haemagglutinin molecule from a highly virulent avian influenza virus. On immunization of chickens and turkeys with the recombinant, protection is afforded against a lethal challenge with either the homologous or a heterologous influenza virus strain.

The matrix (M) protein of influenza A virus, one of the two group-specific internal proteins of the virion, was isolated in a pure form, and its immunogenicity was stable to heating at 100 degrees C for 2 min. Mice immunized with isolated M protein in complete Freund adjuvant and subsequently infected with influenza virus cleared virus more rapidly from their lungs than did unimmunized mice. Despite the rapid clearance of virus, the mice developed pneumonia that was at least as severe as in unimmunized mice. Preliminary studies suggest that the rapid clearance of influenza virus from the lungs of mice immunized with M protein may be initiated by a cell-mediated rather than a humoral response. The mechanism by which a cross-reactive internal virion protein can initiate clearance of the different subtypes of influenza is not clear. Perhaps the M protein is exposed on the surface of the virus-infected cell and is responsible for the cross-reactivity at the cytotoxic T-cell level recently detected between influenza A virus subtypes.

To study the immune response of the chicken to specific influenza proteins, we have constructed a recombinant vaccinia virus containing the hemagglutinin gene of influenza A/Turkey/Ireland/1378/83 (H5N8). In mammalian cell culture the hemagglutinin expressed by this recombinant virus was full-length, cleaved into HA1 and HA2 in the absence of trypsin, and transported to the cell surface, confirming that other virus products are not required for cleavage activation. Chickens inoculated through the wing web with the live recombinant virus produced extremely low levels of hemagglutination-inhibiting or infectivity-neutralizing antibody. However, they were protected from lethal H5 influenza virus challenge. Protection extended to the antigenically distinct virulent H5 viruses, Chicken/Pennsylvania/1370/83 and Chicken/Scotland/59. Chemically bursectomized vaccinated chickens were not protected, whereas normal chickens with very low antibody levels (less than 10) obtained by passive transfer were protected in a dose-dependent fashion. This indicates that despite the low antibody titers induced by vaccination, protection was mediated by antibody.

The A/Hong Kong/1/68 (H3N2) influenza virus which has not been isolated from man for several years, was recently isolated from pigs in Hong Kong. Influenza viruses similar to A/Victoria/3/75, which are currently circulating in man, were also isolated from pigs. Both above-mentioned viruses could be transmitted readily from pig to pig in experimental studies. The isolation of influenza viruses similar to A/Hong Kong/68 from pigs in 1976 suggests that pigs may serve as a potential reservoir for future human pandemics as well as a possible source of genetic information for recombination between human and porcine strains of influenza virus.

Studies on influenza viruses from feral ducks trapped in Canada in August 1976, gave a 26% isolation rate from cloacal samples of juvenile birds. Several different influenza A viruses were isolated, some of which possessed novel hemagglutinin and/or neuraminidase antigens. Influenza A viruses isolated from the rectum of feral ducks replicate in the upper respiratory tract and also in the intestinal tract of feral and domestic ducks. Representative human influenza viruses of the H0N1, H3N2 and Hsw1 N1 subtypes replicate in the upper respiratory tract of ducks but not in the intestinal tract. The A/Hong Kong/68 [H3N2] influenza virus that has not been isolated from man for several years was recently isolated from pigs originating from The People's Republic of China. A/Victoria/3/75-like influenza viruses that are currently circulating in man were also isolated from pigs. Both the A/Hong Kong/68 and the A/Victoria/75-like viruses transmitted readily from pig to pig in experimental studies. The susceptibility of ducks and pigs to infection with human influenza viruses suggests that these animals may play an important role in the ecology of influenza A viruses.

The second phase of a 2-year influenza virus surveillance programme of domestic avian species in Hong Kong (up to October 1977) yielded influenza A virus, Newcastle disease virus, and Hong Kong paramyxovirus, as well as unidentified haemagglutinating agents. These viruses were isolated from the trachea or cloaca of apparently healthy domestic ducks, geese, and chickens originating from China and Hong Kong. Twenty-five combinations of haemagglutinin and neuraminidase surface antigens were identified from the 136 influenza A viruses isolated. Eight of the combinations do not appear to have been previously reported - Hav3Nav2, Hav4Nav2, Hav4Nav4, Hav4Nav5, Hav4Neq1, Hav6Nav4, Hav6Nav6, and Hav9Nav1. The existence of such a diverse pool of influenza virus genetic information may play a role in the emergence of new human pandemic strains.

The role of the neuraminidase in eliciting protection against a lethal influenza A virus [A/Ck/Penn/1370/83 (H5N2)] infection was investigated in chickens. Isolated N2 neuraminidase administered in adjuvant did not prevent infection but did prevent systemic spread of virus and death of chickens. N2 expressed in a recombinant vaccinia virus protected chickens when administered in adjuvant but was less effective when allowed to replicate and produce pox on the chicken's comb. Chickens vaccinated with isolated N2 in adjuvant or with inactivated H5N2 influenza virus were protected from clinical signs and death after challenge with A/Ck/Penn/1370/83 influenza virus. However, these animals were completely susceptible and died of infection with a heterologous subtype (H7N7) of influenza virus. The role of the different antigenic determinants of the N2 NA was investigated in chickens by passive transfer of monoclonal antibodies. Antibodies to antigenic determinants rimming the enzyme active center reduced disease signs in approximately half of the birds but did not significantly reduce virus levels. Antibodies to one of the two independent antigenic determinants that are distant from the enzyme active center were most effective at reducing virus replication and disease signs. This is surprising because antigenic variants could not be selected in vitro with these antibodies and suggests that they may facilitate clearance of virus. Antibodies to the other determinant that is located distally to the enzyme site were ineffective at providing protection.