一株H1N1亚型猪流感病毒的分离鉴定及全基因序列分析

2006年从辽宁省某猪场采集具有流感症状猪鼻拭子共30份,经9~11日龄SPF鸡胚分离,对分离株进行了血凝试验、血凝抑制试验、RT-PCR亚型鉴定,全基因序列比对和接种试验动物试验。结果表明:该毒株具有凝集0.5%鸡红细胞的活性,且与抗猪流感H1标准血清发生血凝抑制反应,RT-PCR分别扩增得到全基因8个片段,利用DNAStar生物学软件进行序列分析,HA基因与GenBank登录的H1~H16中的H1基因序列同源性最高,NA基因与N1~N9中的N1基因序列同源性最高,故该LN株分离毒为猪流感H1N1亚型病毒。全基因序列中,除了M基因不是猪源的,其它基因片段均与国内H1N1亚型猪流感参照株高度同源,推测LN株可能是由类人和类禽谱系的流感病毒与古典猪谱系的流感病毒重排形成的;用该病毒接种试验动物可成功复制出猪流感典型症状。该病毒的分离鉴定及全基因组序列分析为我国进一步调查猪流感的流行规律提供了基础数据。     

Domestic pigs have low susceptibility to H5N1 highly pathogenic avian influenza viruses

Genetic reassortment of H5N1 highly pathogenic avian influenza viruses (HPAI) with currently circulating human influenza A strains is one possibility that could lead to efficient human-to-human transmissibility. Domestic pigs which are susceptible to infection with both human and avian influenza A viruses are one of the natural hosts where such reassortment events could occur. Virological, histological and serological features of H5N1 virus infection in pigs were characterized in this study. Two- to three-week-old domestic piglets were intranasally inoculated with 10(6) EID(50) of A/Vietnam/1203/04 (VN/04), A/chicken/Indonesia/7/03 (Ck/Indo/03), A/Whooper swan/Mongolia/244/05 (WS/Mong/05), and A/Muscovy duck/Vietnam/ 209/05 (MDk/VN/05) viruses. Swine H3N2 and H1N1 viruses were studied as a positive control for swine influenza virus infection. The pathogenicity of the H5N1 HPAI viruses was also characterized in mouse and ferret animal models. Intranasal inoculation of pigs with H5N1 viruses or consumption of infected chicken meat did not result in severe disease. Mild weight loss was seen in pigs inoculated with WS/Mong/05, Ck/Indo/03 H5N1 and H1N1 swine influenza viruses. WS/Mong/05, Ck/Indo/03 and VN/04 viruses were detected in nasal swabs of inoculated pigs mainly on days 1 and 3. Titers of H5N1 viruses in nasal swabs were remarkably lower compared with those of swine influenza viruses. Replication of all four H5N1 viruses in pigs was restricted to the respiratory tract, mainly to the lungs. Titers of H5N1 viruses in the lungs were lower than those of swine viruses. WS/Mong/05 virus was isolated from trachea and tonsils, and MDk/VN/05 virus was isolated from nasal turbinate of infected pigs. Histological examination revealed mild to moderate bronchiolitis and multifocal alveolitis in the lungs of pigs infected with H5N1 viruses, while infection with swine influenza viruses resulted in severe tracheobronchitis and bronchointerstitial pneumonia. Pigs had low susceptibility to infection with H5N1 HPAI viruses. Inoculation of pigs with H5N1 viruses resulted in asymptomatic to mild symptomatic infection restricted to the respiratory tract and tonsils in contrast to mouse and ferrets animal models, where some of the viruses studied were highly pathogenic and replicated systemically.     

Detection and Isolation of Swine Influenza A Virus in Spiked Oral Fluid and Samples from Individually Housed,Experimentally Infected Pigs: Potential Role of Porcine Oral Fluid in Active Influenza A Virus Surveillance in Swine

Background

The lack of seasonality of swine influenza A virus (swIAV) in combination with the capacity of swine to harbor a large number of co-circulating IAV lineages, resulting in the risk for the emergence of influenza viruses with pandemic potential, stress the importance of swIAV surveillance. To date, active surveillance of swIAV worldwide is barely done because of the short detection period in nasal swab samples. Therefore, more sensitive diagnostic methods to monitor circulating virus strains are requisite.

Methods

qRT-PCR and virus isolations were performed on oral fluid and nasal swabs collected from individually housed pigs that were infected sequentially with H1N1 and H3N2 swIAV strains. The same methods were also applied to oral fluid samples spiked with H1N1 to study the influence of conservation time and temperature on swIAV infectivity and detectability in porcine oral fluid.

Results

All swIAV infected animals were found qRT-PCR positive in both nasal swabs and oral fluid. However, swIAV could be detected for a longer period in oral fluid than in nasal swabs. Despite the high detectability of swIAV in oral fluid, virus isolation from oral fluid collected from infected pigs was rare. These results are supported by laboratory studies showing that the PCR detectability of swIAV remains unaltered during a 24 h incubation period in oral fluid, while swIAV infectivity drops dramatically immediately upon contact with oral fluid (3 log titer reduction) and gets lost after 24 h conservation in oral fluid at ambient temperature.

Conclusions

Our data indicate that porcine oral fluid has the potential to replace nasal swabs for molecular diagnostic purposes. The difficulty to isolate swIAV from oral fluid could pose a drawback for its use in active surveillance programs.     

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.     

Evaluation of the ESPLINE INFLUENZA A&B-N Kit for the diagnosis of avian and swine influenza

The ESPLINE INFLUENZA A&B-N kit was evaluated for its applicability to the rapid diagnosis of influenza in chickens and pigs. The kit specifically detected viral antigens in tracheal swabs and tissue homogenates of the trachea, liver, spleen, and colon of chickens inoculated with a highly pathogenic avian influenza virus strain, A/chicken/Yamaguchi/7/04 (H5N1), at 48 hr post-inoculation (p.i.) as well as in the tracheal and cloacal swabs and tissue homogenates of dead chickens. For those infected with a low pathogenic strain, A/chicken/aq-Y-55/01 (H9N2), antigens were detected only in the samples from tracheal swabs and organs 1-4 days p.i. The kit also detected viral antigens in the nasal swabs of miniature pigs infected with swine and avian influenza viruses. The kit was found to be sensitive and specific enough for the rapid diagnosis of infections of influenza A virus in chickens and pigs.     

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.     

Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.     

Recently emerged swine influenza A virus (H2N3) causes severe pneumonia in Cynomolgus macaques

The triple reassortant H2N3 virus isolated from diseased pigs in the United States in 2006 is pathogenic for certain mammals without prior adaptation and transmits among swine and ferrets. Adaptation, in the H2 hemagglutinin derived from an avian virus, includes the ability to bind to the mammalian receptor, a significant prerequisite for infection of mammals, in particular humans, which poses a big concern for public health. Here we investigated the pathogenic potential of swine H2N3 in Cynomolgus macaques, a surrogate model for human influenza infection. In contrast to human H2N2 virus, which served as a control and largely caused mild pneumonia similar to seasonal influenza A viruses, the swine H2N3 virus was more pathogenic causing severe pneumonia in nonhuman primates. Both viruses replicated in the entire respiratory tract, but only swine H2N3 could be isolated from lung tissue on day 6 post infection. All animals cleared the infection whereas swine H2N3 infected macaques still presented with pathologic changes indicative of chronic pneumonia at day 14 post infection. Swine H2N3 virus was also detected to significantly higher titers in nasal and oral swabs indicating the potential for animal-to-animal transmission. Plasma levels of IL-6, IL-8, MCP-1 and IFNγ were significantly increased in swine H2N3 compared to human H2N2 infected animals supporting the previously published notion of increased IL-6 levels being a potential marker for severe influenza infections. In conclusion, the swine H2N3 virus represents a threat to humans with the potential for causing a larger outbreak in a non-immune or partially immune population. Furthermore, surveillance efforts in farmed pig populations need to become an integral part of any epidemic and pandemic influenza preparedness.     

Spatial dynamics of human-origin H1 influenza A virus in North American swine

The emergence and rapid global spread of the swine-origin H1N1/09 pandemic influenza A virus in humans underscores the importance of swine populations as reservoirs for genetically diverse influenza viruses with the potential to infect humans. However, despite their significance for animal and human health, relatively little is known about the phylogeography of swine influenza viruses in the United States. This study utilizes an expansive data set of hemagglutinin (HA1) sequences (n = 1516) from swine influenza viruses collected in North America during the period 2003-2010. With these data we investigate the spatial dissemination of a novel influenza virus of the H1 subtype that was introduced into the North American swine population via two separate human-to-swine transmission events around 2003. Bayesian phylogeographic analysis reveals that the spatial dissemination of this influenza virus in the US swine population follows long-distance swine movements from the Southern US to the Midwest, a corn-rich commercial center that imports millions of swine annually. Hence, multiple genetically diverse influenza viruses are introduced and co-circulate in the Midwest, providing the opportunity for genomic reassortment. Overall, the Midwest serves primarily as an ecological sink for swine influenza in the US, with sources of virus genetic diversity instead located in the Southeast (mainly North Carolina) and South-central (mainly Oklahoma) regions. Understanding the importance of long-distance pig transportation in the evolution and spatial dissemination of the influenza virus in swine may inform future strategies for the surveillance and control of influenza, and perhaps other swine pathogens.     

Novel reassortment of Eurasian avian-like and pandemic/2009 influenza viruses in swine: infectious potential for humans

Pigs are considered to be intermediate hosts and "mixing vessels," facilitating the genesis of pandemic influenza viruses, as demonstrated by the emergence of the 2009 H1N1 pandemic (pdm/09) virus. The prevalence and repeated introduction of the pdm/09 virus into pigs raises the possibility of generating novel swine influenza viruses with the potential to infect humans. To address this, an active influenza surveillance program was conducted with slaughtered pigs in abattoirs in southern China. Over 50% of the pigs tested were found to be seropositive for one or more H1 influenza viruses, most commonly pdm/09-like viruses. Out of 36 virus isolates detected, one group of novel reassortants had Eurasian avian-like swine H1N1 surface genes and pdm/09 internal genes. Animal experiments showed that this virus transmitted effectively from pig to pig and from pig to ferret, and it could also replicate in ex vivo human lung tissue. Immunization against the 2009 pandemic virus gave only partial protection to ferrets. The continuing prevalence of the pdm/09 virus in pigs could lead to the genesis of novel swine reassortant viruses with the potential to infect humans.     

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.     

Live Attenuated Influenza Vaccine Provides Superior Protection from Heterologous Infection in Pigs with Maternal Antibodies without Inducing Vaccine-Associated Enhanced Respiratory Disease

Control of swine influenza A virus (IAV) in the United States is hindered because inactivated vaccines do not provide robust cross-protection against the multiple antigenic variants cocirculating in the field. Vaccine efficacy can be limited further for vaccines administered to young pigs that possess maternally derived immunity. We previously demonstrated that a recombinant A/sw/Texas/4199-2/1998 (TX98) (H3N2) virus expressing a truncated NS1 protein is attenuated in swine and has potential for use as an intranasal live attenuated influenza virus (LAIV) vaccine. In the present study, we compared 1 dose of intranasal LAIV with 2 intramuscular doses of TX98 whole inactivated virus (WIV) with adjuvant in weanling pigs with and without TX98-specific maternally derived antibodies (MDA). Pigs were subsequently challenged with wild-type homologous TX98 H3N2 virus or with an antigenic variant, A/sw/Colorado/23619/1999 (CO99) (H3N2). In the absence of MDA, both vaccines protected against homologous TX98 and heterologous CO99 shedding, although the LAIV elicited lower hemagglutination inhibition (HI) antibody titers in serum. The efficacy of both vaccines was reduced by the presence of MDA; however, WIV vaccination of MDA-positive pigs led to dramatically enhanced pneumonia following heterologous challenge, a phenomenon known as vaccine-associated enhanced respiratory disease (VAERD). A single dose of LAIV administered to MDA-positive pigs still provided partial protection from CO99 and may be a safer vaccine for young pigs under field conditions, where dams are routinely vaccinated and diverse IAV strains are in circulation. These results have implications not only for pigs but also for other influenza virus host species.     

Vaccination of pigs against swine influenza viruses by using an NS1-truncated modified live-virus vaccine

Swine influenza viruses (SIV) naturally infect pigs and can be transmitted to humans. In the pig, genetic reassortment to create novel influenza subtypes by mixing avian, human, and swine influenza viruses is possible. An SIV vaccine inducing cross-protective immunity between different subtypes and strains circulating in pigs is highly desirable. Previously, we have shown that an H3N2 SIV (A/swine/Texas/4199-2/98 [TX98]) containing a deleted NS1 gene expressing a truncated NS1 protein of 126 amino acids, NS1black triangle126, was attenuated in swine. In this study, 4-week-old pigs were vaccinated with the TX98 NS1black triangle126 modified live virus (MLV). Ten days after boosting, pigs were challenged with wild-type homologous H3N2 or heterosubtypic H1N1 SIV and sacrificed 5 days later. The MLV was highly attenuated and completely protected against challenge with the homologous virus. Vaccinated pigs challenged with the heterosubtypic H1N1 virus demonstrated macroscopic lung lesions similar to those of the unvaccinated H1N1 control pigs. Remarkably, vaccinated pigs challenged with the H1N1 SIV had significantly lower microscopic lung lesions and less virus shedding from the respiratory tract than did unvaccinated, H1N1-challenged pigs. All vaccinated pigs developed significant levels of hemagglutination inhibition and enzyme-linked immunosorbent assay titers in serum and mucosal immunoglobulin A antibodies against H3N2 SIV antigens. Vaccinated pigs were seronegative for NS1, indicating the potential use of the TX98 NS1black triangle126 MLV as a vaccine to differentiate infected from vaccinated animals.     

Clinical Profiles Associated with Influenza Disease in the Ferret Model

Influenza A viruses continue to pose a threat to human health; thus, various vaccines and prophylaxis continue to be developed. Testing of these products requires various animal models including mice, guinea pigs, and ferrets. However, because ferrets are naturally susceptible to infection with human influenza viruses and because the disease state resembles that of human influenza, these animals have been widely used as a model to study influenza virus pathogenesis. In this report, a statistical analysis was performed to evaluate data involving 269 ferrets infected with seasonal influenza, swine influenza, and highly pathogenic avian influenza (HPAI) from 16 different studies over a five year period. The aim of the analyses was to better qualify the ferret model by identifying relationships among important animal model parameters (endpoints) and variables of interest, which include survival, time-to-death, changes in body temperature and weight, and nasal wash samples containing virus, in addition to significant changes from baseline in selected hematology and clinical chemistry parameters. The results demonstrate that a disease clinical profile, consisting of various changes in the biological parameters tested, is associated with various influenza A infections in ferrets. Additionally, the analysis yielded correlates of protection associated with HPAI disease in ferrets. In all, the results from this study further validate the use of the ferret as a model to study influenza A pathology and to evaluate product efficacy.     

Evaluation of sampling tools for environmental sampling of bacterial endospores from porous and nonporous surfaces

Aims: Having and executing a well-defined and validated sampling protocol is critical following a purposeful release of a biological agent for response and recovery activities, for clinical and epidemiological analysis and for forensic purposes. The objective of this study was to address the need for validated sampling and analysis methods called out by the General Accounting Office and others to systematically compare the collection efficiency of various swabs and wipes for collection of bacterial endospores from five different surfaces, both porous and nonporous. This study was also designed to test the collection and extraction solutions used for endospore recovery from swabs and wipes. Methods and Results: Eight collection tools, five swabs and three wipes, were used. Three collection/preservation solutions were evaluated: an ink jet aerosol generator was used to apply Bacillus subtilis endospores to five porous and nonporous surfaces. The collection efficiencies of the swabs and wipes were compared using a statistical multiple comparison analysis. Conclusions: The ScottPure^® wipe had the highest collection efficiency and phosphate-buffered saline (PBST) with 0·3% Tween was the best collection solution of those tested. Significance and Impact of the Study: Validated sampling for potential biological warfare is of significant importance and this study answered some relevant questions.