Significant rising antibody titres to influenza A are associated with an acute reduction in milk yield in cattle

Sporadic cases of an acute fall in milk production, "milk drop", were investigated in a Holstein Friesian dairy herd in Devon. The investigation was a case control study with two controls per case. Paired blood samples demonstrated that rising antibody titres to human influenza A/England/333/80 (H1N1) and human influenza A/Eng/427/88 (H3N2) were associated with an acute fall in milk production. Rising titres to bovine respiratory syncytial virus (BRSV), bovine virus diarrhoea virus (BVD), infectious bovine rhinotracheitis (IBR) and parainfluenza virus 3 (PI3) were not associated with an acute fall in milk production. Cases with rises in antibody to influenza A had significantly higher respiratory scores and rectal temperatures than their controls. The mean loss of milk production for the cases with rises in antibody to influenza A compared to their controls was 159.9L. This study provides further evidence that influenza A persists in cattle and causes clinical disease.     

Seroprevalence of porcine and human influenza A virus antibodies in pigs between 1986 and 1988 in Hassia

1,268 sera collected from slaughtered pigs in Hassia (FRG) from 1986 to 1988 were tested for antibodies against porcine and human influenza A virus strains using the single radial haemolysis test (SRHT). Antibodies against the porcine strains (subtype H1N1) A/Swine/Arnsberg/1/81, A/Swine/Iowa/15/30 and A/New Jersey/7/76 were detected in 411 (32.4%), 318 (25.1%) and 304 (24.0%) of sera, respectively. Up to 1988 a slight increase (10%) in the seroprevalence to A/Swine/Arnsberg/1/81 was noticed, whereas the results obtained with the other strains showed little variation. Antibodies against the human H1N1 strain A/Singapore/6/86 were only found in sera collected 1987 and 1988 in rates of 1.6% and 3.0%. Serological indication of infections with the human H3N2 strains A/Victoria/1/75, A/Hong Kong/1/68 and A/Philippines/2/82 could be shown in 286 (22.6%), 178 (14.4%) and 135 (10.6%) of the serum samples. Within the three year period the rate of sera positive for antibodies against A/Philippines/2/82 increased from 6.5% to 23.0%, whereas no variation in the rates were found using the other H3N2 strains. Antibodies simultaneously against porcine (H1N1) and human (H3N2) virus strains were detected in 9.9% of all sera tested.     

Outbreaks of classical swine influenza in pigs in England in 1986

Serum samples from pig herds in Great Britain have been examined for antibodies to influenza virus since 1968. Antibodies to H3N2 virus strains have been found since 1968 and the serological data presented here suggests that H3N2 virus strains continue to persist in the pig population. An outbreak of acute respiratory disease occurred in a 400-sow unit. The outbreak was characterised by coughing, anorexia, fever, inappetence and loss of condition. The gilts and weaners were affected and the morbidity approached 100 per cent. An influenza A virus designated A/Swine/Weybridge/117316/86 (H1N1) was isolated from the herd and 28 paired serum samples from the affected animals showed increases in the haemagglutination inhibition titres to this isolate. Haemagglutinin and neuraminidase characterisation indicated that the virus is similar to H1N1 viruses isolated recently from pigs in Europe. A total of 91 herds experiencing respiratory disease were investigated, of which 42 gave positive reactions in the haemagglutination inhibition test. Antibodies to A/Port Chalmers/1/73 (H3N2) were also detected in some of the herds but it is not known whether this strain plays any role in the current respiratory disease problems in pigs.     

Influenza A viruses: epidemiologic study in fatteners in Spain (1987-89).

2,979 sera were collected from slaughtered swine in two geographic areas of Spain from 1987 to 1989. They were tested for antibodies against an H1N1- and H3N2-influenza virus by haemagglutination-inhibition tests (HI). The percentage of positive sera was higher in area I (78%-69.2%) than in area II (63.1%-60.4%) for both viruses respectively. The coexistence of high titres to both H1N1- and H3N2-influenza virus became apparent in cold months simultaneously in each area, although influenza viruses circulated in the Spanish swine population for two years. Also this study suggests the possible circulation of A/Texas/1/77-like strains in Spain, results which have not been reported before.     

The prevalence of antibodies to influenza virus and respiratory coronavirus in fattening pigs in Spain

The presence of antibodies to two influenza viruses of the type A (H1N1 and H3N2) and to a porcine respiratory coronavirus was investigated in a study lasting a year. 735 blood serum samples were collected from 79 closed pig fattening farms in the province Segovia (Spain). Hemagglutination inhibition was used with influenza viruses. The percentage of positive results was 78.5% and 62.5% respectively for the serotypes H1N1 and H3N2. A clear reduction in the spread of antibodies was observed in the autumn. The ELISA technique was used with the porcine respiratory coronavirus. As antigen we used the antigenically related transmissible porcine gastroenteritis virus. Using this technique 87% of the sera were positive. Some of these sera with representative ELISA values were confirmed by means of serum neutralisation and radioimmune precipitation of the viral proteins. The incidence of these antibodies remained unchanged over the whole year of the investigation.     

Serological surveillance reveals widespread influenza A H7 and H9 subtypes among chicken flocks in Egypt

Multiple avian influenza viruses’ subtypes are circulating worldwide possessing serious threat to human populations and considered key contributors to the emergence of human influenza pandemics. This study aimed to identify the potential existence of H7 and H9 avian influenza infections circulating among chicken flocks in Egypt. Serum samples were collected from chicken flocks that experienced respiratory distresses and/or variable mortality rates. H7 and H9 virus infections were screened by haemagglutination inhibition assay using chicken erythrocytes. Serum samples were collected from 9 broiler, 12 breeder and 18 layer flocks. Out of 1,225 examined sera, 417 (34 %) from 14 flocks and 605 (49.4 %) from 21 flocks were found positive for H7 and H9, respectively. Prevalence of both H7 and H9 antibodies were higher in layer followed by breeder then broiler flocks. Special consideration should be paid to control influenza viruses in Egypt, as pandemic influenza strains may develop unnoticed given the presence of subclinical infections, and the possibility of re-assortment with the prevailing endemic H5N1 virus strains in Egypt do exist.     

Replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses

Precision-cut lung slices of pigs were infected with five swine influenza A viruses of different subtypes (A/sw/Potsdam/15/1981 H1N1, A/sw/Bad Griesbach/IDT5604/2006 H1N1, A/sw/Bakum/1832/2000 H1N2, A/sw/Damme/IDT5673/2006 H3N2, A/sw/Herford/IDT5932/2007 H3N2). The viruses were able to infect ciliated and mucus-producing cells. The infection of well-differentiated respiratory epithelial cells by swine influenza A viruses was analyzed with respect to the kinetics of virus release into the supernatant. The highest titres were determined for H3N2/2006 and H3N2/2007 viruses. H1N1/1981 and H1N2/2000 viruses replicated somewhat slower than the H3N2 viruses whereas a H1N1 strain from 2006 multiplied at significantly lower titres than the other strains. Regarding their ability to induce a ciliostatic effect, the two H3N2 strains were found to be most virulent. H1N1/1981 and H1N2/2000 were somewhat less virulent with respect to their effect on ciliary activity. The lowest ciliostatic effect was observed with H1N1/2006. In order to investigate whether this finding is associated with a corresponding virulence in the host, pigs were infected experimentally with H3N2/2006, H1N2/2000, H1N1/1981 and H1N1/2006 viruses. The H1N1/2006 virus was significantly less virulent than the other viruses in pigs which was in agreement with the results obtained by the in vitro-studies. These findings offer the possibility to develop an ex vivo-system that is able to assess virulence of swine influenza A viruses.     

Serological evidence for exposure to avian influenza viruses within poultry workers in southern China

The risk of infection with avian influenza viruses for poultry workers is relatively unknown in China, and study results are often biased by the notification of only the severe human cases. Protein microarray was used to detect binding antibodies to 13 different haemagglutinin (HA1‐part) antigens of avian influenza A(H5N1), A(H7N7), A(H7N9) and A(H9N2) viruses, in serum samples from poultry workers and healthy blood donors collected in the course of 3 years in Guangdong Province, China. Significantly higher antibody titre levels were detected in poultry workers when compared to blood donors for the most recent H5 and H9 strains tested. These differences were most pronounced in younger age groups for antigens from older strains, but were observed in all age groups for the recent H5 and H9 antigens. For the H7 strains tested, only poultry workers from two retail live poultry markets had significantly higher antibody titres compared to blood donors.     

Antibody responses of swine to type A influenza viruses in the most recent several years.

A serological survey was conducted on 4,080 swine sera collected for the years 1985-90. The swine sera positive to A/New Jersey/8/76 (swine type H1N1) strain were observed in annual (10-20%) and monthly (20-40%) incidences during the observation period except for occasional months. Antibodies to recent human H1N1 viruses in swine were recognized in relation to the human H1N1 influenza epidemics. Antibody responses of swine to human H3N2 strains appeared irrespective of human epidemics with the virus in the years 1985-87. However, in 1988 almost no antibodies to three human H3N2 isolates of 1983-88 were observed for this year except a few months though the human epidemic occurred in the area. Although in 1989-90 many swine had antibodies to the three strains in the percentage of 3 to 35, no antibody to the latest isolate, A/Hokkaido/20/89 (H3N2), was found for almost all the months of both years. These findings differed markedly from the possible relationship between the prevalence of H3N2 virus-antibodies in swine and the human influenza epidemics, which were described previously in many reports including our studies.     

Analysis of the quality of protection induced by a porcine influenza A vaccine to challenge with an H3N2 virus

Antigenic drift of swine influenza A (H3N2) viruses away from the human A/Port Chalmers/1/73 (H3N2) strain, used in current commercial swine influenza vaccines, has been demonstrated in The Netherlands and Belgium. Therefore, replacement of this human strain by a more recent swine H3N2 isolate has to be considered. In this study, the efficacy of a current commercial swine influenza vaccine to protect pigs against a recent Dutch field strain (A/Sw/Oedenrode/96) was assessed. To evaluate the level of protection induced by the vaccine it was compared with the optimal protection induced by a previous homologous infection. Development of fever, virus excretion, and viral transmission to unchallenged group mates were determined to evaluate protection. The vaccine appeared efficacious in the experiment because it was able to prevent fever and virus transmission to the unchallenged group mates. Nevertheless, the protection conferred by the vaccine was sub-optimal because vaccinated pigs excreted influenza virus for a short period of time after challenge, whereas naturally immune pigs appeared completely protected. The immune response was monitored, to investigate why the vaccine conferred a sub-optimal protection. The haemagglutination inhibiting and virus neutralising antibody responses in sera, the nucleoprotein-specific IgM, IgG, and IgA antibody responses in sera and nasal secretions and the influenza-specific lymphoproliferation responses in the blood were studied. Vaccinated pigs developed the same or higher serum haemagglutination inhibiting, virus neutralising, and nucleoprotein-specific IgG antibody titres as infected pigs but lower nasal IgA titres and lymphoproliferation responses. The lower mucosal and cell-mediated immune responses may explain why protection after vaccination was sub-optimal.     

Influenza surveillance in birds in Italian wetlands (1992-1998): is there a host restricted circulation of influenza viruses in sympatric ducks and coots?

We report the results of a 6-year serological and virological monitoring performed in ducks and coots in Italy, in order to assess the degree of influenza A virus circulation in these birds during wintering. A total of 1039 sera collected from 1992 to 1998 was screened by a double antibody sandwich blocking ELISA (NP-ELISA): seroprevalence of antibodies to influenza A viruses was significantly higher in ducks compared to coots (52.2% vs. 7.1%, respectively). The hemagglutination-inhibition (HI) assay, performed on NP-ELISA positive sera, showed that 16.9% of these duck sera and 33.3% of these coot sera had antibodies to at least one influenza virus HA subtype: ducks showed HI antibodies against most of the HA subtypes, except for the H3, H4, H7, and H12; coots were seropositive to the H3 and H10 subtypes, only. From 1993 to 1998, 22 virus strains were obtained from 802 cloacal swabs, with an overall virus isolation frequency of 2.7%. Viruses belonging to the H1N1 subtype were by far the most commonly circulating strains (18/22) and were isolated mainly from ducks (17/18). The remaining viruses were representative of the H10N8, H5N2 and H3N8 subtypes. Our data indicate some differences between influenza A virus circulation in sympatric ducks and coots and a significant antigenic diversity between some reference strains and viruses recently isolated in Italy.     

Differentiation of matrix proteins of influenza A viruses using enzyme immunoassay]

Matrix protein is known as a type-specific structural protein of influenza viruses. An attempt has been made to find out whether or not strain-specific components could be detected from matrix protein, in addition to its type-specific antigen determinants. The technique of enzyme immune assay was chosen as the optional method to differentiate between matrix proteins of various influenza-A viruses. Antigen titration was undertaken of several matrix proteins, using two specific anti-matrix-protein sera in each case. Information regarding serological relationships between the tested matrix proteins of various influenza-A viruses was obtained from a quotient between the titres of one antigen, on the one hand, and the two anti-matrix-protein sera used in titration, on the other. Two matrix protein sub-types were established in the context of the influenza-A viruses tested. Sub-type M1 was attributable to older strains (A/PR/8 and A/FM/1), whereas the matrix protein of sub-type M2 was found to be present in more recent strains (A/Hongkong and A/Port Chalmers).     

Seroepidemiological evidence of avian H4, H5, and H9 influenza A virus transmission to pigs in southeastern China

Pig serum samples collected in southeastern China were examined for antibodies to influenza A viruses. Since the hemagglutination inhibition (HI) test does not accurately detect antibodies to the hemagglutinins (HAs) of "avian" influenza viruses, we utilized the neutralization (NT) test to detect subtype-specific antibodies to the HA of avian viruses in pig sera. Neutralizing antibodies to H1, H3, H4, and H5 influenza viruses were detected in the serum samples collected in 1977-1982 and 1998, suggesting that pigs in China have been sporadically infected with avian H4 and H5 viruses in addition to swine and human H1 and H3 viruses. Antibodies to H9 virus, on the other hand, were found only in the sera collected in 1998, not in those collected in 1977-1982, correlating with the recent spread in poultry and subsequent isolation of H9N2 viruses from pigs and humans in 1998. The present results indicate that avian influenza viruses have been transmitted to pig populations in southeastern China.     

Virological Surveillance and Preliminary Antigenic Characterization of Influenza Viruses in Pigs in Five European Countries from 2006 to 2008

This study presents the results of the virological surveillance for swine influenza viruses (SIVs) in Belgium, UK, Italy, France and Spain from 2006 to 2008. Our major aims were to clarify the occurrence of the three SIV subtypes – H1N1, H3N2 and H1N2 – at regional levels, to identify novel reassortant viruses and to antigenically compare SIVs with human H1N1 and H3N2 influenza viruses. Lung tissue and/or nasal swabs from outbreaks of acute respiratory disease in pigs were investigated by virus isolation. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined using standard methods. Of the total 169 viruses, 81 were classified as ‘avian‐like’ H1N1, 36 as human‐like H3N2 and 47 as human‐like H1N2. Only five novel reassortant viruses were identified: two H1N1 viruses had a human‐like HA and three H1N2 viruses an avian‐like HA. All three SIV subtypes were detected in Belgium, Italy and Spain, while only H1N1 and H1N2 viruses were found in UK and Northwestern France. Cross‐hemagglutination inhibition (HI) tests with hyperimmune sera against selected older and recent human influenza viruses showed a strong antigenic relationship between human H1N1 and H3N2 viruses from the 1980s and H1N2 and H3N2 human‐like SIVs, confirming their common origin. However, antisera against human viruses isolated during the last decade did not react with currently circulating H1 or H3 SIVs, suggesting that especially young people may be, to some degree, susceptible to SIV infections.     

猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法的建立

对我国分离到的猪流感病毒和GenBank数据库中已有的猪流感病毒H1N1、H1N2和H3N2亚型毒株的HA、NA基因核苷酸序列进行分析,分别选出各个病毒亚型HA和NA基因中高度保守且特异的核苷酸区域,设计扩增猪流感病毒H1和H3、N1和N2亚型的2套多重PCR特异性引物,建立了猪流感H1N1、H1N2和H3N2亚型病毒多重RT-PCR诊断方法。采用该方法对H1N1、H1N2、H3N2亚型猪流感病毒标准参考株进行RT-PCR检测,结果均呈阳性,对扩增得到的片段进行序列测定和BLAST比较,表明为目的基因片段。其它几种常见猪病病毒和其它亚型猪流感病毒的RT-PCR扩增结果都呈阴性。对107EID50/0.1mL病毒进行稀释,提取RNA进行敏感性试验,RT-PCR最少可检测到102EID50的病毒量核酸。对40份阳性临床样品的检测结果是H1N1、H1N2和H3N2亚型分别为16份、1份和20份,其它3份样品同时含有H1N1和H3N2亚型猪流感病毒,和鸡胚分离病毒结果100%一致。试验证明建立的猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法是一种特异敏感的诊断方法,可用于临床样品的早期快速诊断和分型。     

Genetic diversity of H9N2 influenza viruses from pigs in China: a potential threat to human health?

Pandemic strains of influenza A virus might arise by genetic reassortment between viruses from different hosts. Pigs are susceptible to both human and avian influenza viruses and 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 summarize and report for the first time the coexistence of 10 (A-J) genotypes in pigs in China by analyzing the eight genes of 28 swine H9N2 viruses isolated in China from 1998 to 2007. Swine H9N2 viruses in genotype A and B were completely derived from Y280-like and Shanghai/F/98-like viruses, respectively, which indicated avian-to-pig interspecies transmission of H9N2 viruses did exist in China. The other eight genotype (C-J) viruses might be double-reassortant viruses, in which six genotype (E-J) viruses possessed 1-4 H5-like gene segments indicating they were reassortants of H9 and H5 viruses. In conclusion, genetic diversity of H9N2 influenza viruses from pigs in China provides further evidence that avian to pig interspecies transmission of H9N2 viruses did occur and might result in the generation of new reassortant viruses by genetic reassortment with swine H1N1, H1N2 and H3N2 influenza viruses, therefore, these swine H9N2 influenza viruses might be a potential threat to human health and continuing to carry out swine influenza virus surveillance in China is of great significance.     

Serological Evidence of Pandemic H1N1 Influenza Virus Infections in Greek Swine

The introduction of the 2009 pandemic H1N1 (pH1N1) influenza virus in pigs changed the epidemiology of influenza A viruses (IAVs) in swine in Europe and the rest of the world. Previously, three IAV subtypes were found in the European pig population: an avian‐like H1N1 and two reassortant H1N2 and H3N2 viruses with human‐origin haemagglutinin (HA) and neuraminidase proteins and internal genes of avian decent. These viruses pose antigenically distinct HAs, which allow the retrospective diagnosis of infection in serological investigations. However, cross‐reactions between the HA of pH1N1 and the HAs of the other circulating H1 IAVs complicate serological diagnosis. The prevalence of IAVs in Greek swine has been poorly investigated. In this study, we examined and compared haemagglutination inhibition (HI) antibody titres against previously established IAVs and pH1N1 in 908 swine sera from 88 herds, collected before and after the 2009 pandemic. While we confirmed the historic presence of the three IAVs established in European swine, we also found that 4% of the pig sera examined after 2009 had HI antibodies only against the pH1N1 virus. Our results indicate that pH1N1 is circulating in Greek pigs and stress out the importance of a vigorous virological surveillance programme.     

供港猪群流感病毒的分离鉴定及流行病学调查

为了对供港猪群中的猪流感流行情况进行分析,从华南地区供港猪群中用无菌棉拭子采集鼻腔粘液样品,采用鸡胚接种方法,从供港猪群中分离出了2株不同亚型的猪流感病毒株,经国家流感中心鉴定分别为H1N1和H3N2亚型。本研究设计了猪流感常见亚型的HA和NA分型特异性引物,建立了猪流感型特异性RT-PCR检测方法;对分离鉴定的2株猪流感病毒和禽流感H5N1 HI检测抗原进行了RT-PCR检测,并对其部分HA和NA基因进行克隆测序分析。对供港猪群的血清检测结果表明:供港猪群中H1N1和H3N2亚型抗体阳性率分别为26.87%、38.26%,禽流感H5N1和H9N2亚型抗体阳性率均为0%。     

Different evolutionary trends of swine H1N2 influenza viruses in Italy compared to European viruses

European H1N2 swine influenza viruses (EU H1N2SIVs) arose from multiple reassortment events among human H1N1, human H3N2, and avian influenza viruses. We investigated the evolutionary dynamics of 53 Italian H1N2 strains by comparing them with EU H1N2 SIVs. Hemagglutinin (HA) phylogeny revealed Italian strains fell into four groups: Group A and B (41 strains) had a human H1 similar to EU H1N2SIVs, which probably originated in 1986. However Group B (38 strains) formed a subgroup that had a two-amino acid deletion at positions 146/147 in HA. Group C (11 strains) contained an avian H1 that probably originated in 1996, and Group D (1 strain) had an H1 characteristic of the 2009 pandemic strain. Neuraminidase (NA) phylogeny suggested a series of genomic reassortments had occurred. Group A had an N2 that originated from human H3N2 in the late 1970s. Group B had different human N2 that most likely arose from a reassortment with the more recent human H3N2 virus, which probably occurred in 2000. Group C had an avian-like H1 combined with an N2 gene from one of EU H1N2SIVs, EU H3N2SIVs or Human H3N2. Group D was part of the EU H3N2SIVs clade. Although selection pressure for HA and NA was low, several positively selected sites were identified in both proteins, some of which were antigenic, suggesting selection influenced the evolution of SIV. The data highlight different evolutionary trends between European viruses and currently circulating Italian B strains and show the establishment of reassortant strains involving human viruses in Italian pigs.     

Neutralising antibody to herpesviruses dervied from wildebeest and hartebeest in wild animals in East Africa.

The sera of 728 game animals, collected in East Africa, were tested for neutralising antibody to a strain (WC11) of wildebeest herpesvirus, which is an important cause of malignant catarrhal fever of cattle. In addition, 290 of these sera were tested for neutralising activity against a closely related herpesvirus (K/30) from hartebeest (Alcelaphus sp.). Antibody was frequently present in three species of the subfamily Alcelaphinae (wildebest, hartebeest and topi) and one of the subfamily Hippotraginae (oryx). No activity was found in the sera of nine other species of four additional subfamilies. The proportion of hartebeest sera positive for WC11 virus (60 per cent) was not significantly different from that neutralising the K/30 isolate (77 per cent) and antibody titres against the two agents did not differ significantly. Of 62 topi sera 40 per cent neutralised WC11 virus, while all of three oryx sera were positive. It is suggested that the antibody detected in hartebeest, topi and oryx was induced by infection with viruses related to, but not identical with, the WC11 and K/30 strains. Some characteristics of the latter indicated that it was not the usual herpesvirus of hartebeest and may have been derived from wildebeest. It is proposed that the group of viruses involved should be provisionally designated as 'alcelaphine herpesviruses' in order to separate them from the rest of the 'bovid' herpesviruses, a name proposed by the Herpesvirus Sbucommittee of the International Committee on the Nomenclature of Viruses.