The first detection of influenza in the Finnish pig population: a retrospective study

Background

Swine influenza is an infectious acute respiratory disease of pigs caused by influenza A virus. We investigated the time of entry of swine influenza into the Finnish pig population. We also describe the molecular detection of two types of influenza A (H1N1) viruses in porcine samples submitted in 2009 and 2010.This retrospective study was based on three categories of samples: blood samples collected for disease monitoring from pigs at major slaughterhouses from 2007 to 2009; blood samples from pigs in farms with a special health status taken in 2008 and 2009; and diagnostic blood samples from pigs in farms with clinical signs of respiratory disease in 2008 and 2009. The blood samples were tested for influenza A antibodies with an antibody ELISA. Positive samples were further analyzed for H1N1, H3N2, and H1N2 antibodies with a hemagglutination inhibition test. Diagnostic samples for virus detection were subjected to influenza A M-gene-specific real-time RT-PCR and to pandemic influenza A H1N1-specific real-time RT-PCR. Positive samples were further analyzed with RT-PCRs designed for this purpose, and the PCR products were sequenced and sequences analyzed phylogenetically.

Results

In the blood samples from pigs in special health class farms producing replacement animals and in diagnostic blood samples, the first serologically positive samples originated from the period July–August 2008. In samples collected for disease monitoring, < 0.1%, 0% and 16% were positive for antibodies against influenza A H1N1 in the HI test in 2007, 2008, and 2009, respectively. Swine influenza A virus of avian-like H1N1 was first detected in diagnostic samples in February 2009. In 2009 and 2010, the avian-like H1N1 virus was detected on 12 and two farms, respectively. The pandemic H1N1 virus (A(H1N1)pdm09) was detected on one pig farm in 2009 and on two farms in 2010.

Conclusions

Based on our study, swine influenza of avian-like H1N1 virus was introduced into the Finnish pig population in 2008 and A(H1N1)pdm09 virus in 2009. The source of avian-like H1N1 infection could not be determined. Cases of pandemic H1N1 in pigs coincided with the period when the A(H1N1)pdm09 virus was spread in humans in Finland.     

Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus

Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1, H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until this moment there are no reports of IAV infection in wild boars or in captive wild boars populations. Herein the occurrence of IAV in captive wild boars with the presence of lung consolidation lesions during slaughter was investigated. Lung samples were screened by RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR. Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using Illumina's genome analyzer platform (MiSeq). The genomic analysis revealed that the HA and NA genes clustered with IAVs of the human lineage and the six internal genes were derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to monitor IAV evolution in Suidae populations.     

Genetic analysis of human and swine influenza A viruses isolated in Northern Italy during 2010–2015

Influenza A virus (IAV) infection in swine plays an important role in the ecology of influenza viruses. The emergence of new IAVs comes through different mechanisms, with the genetic reassortment of genes between influenza viruses, also originating from different species, being common. We performed a genetic analysis on 179 IAV isolates from humans (n. 75) and pigs (n. 104) collected in Northern Italy between 2010 and 2015, to monitor the genetic exchange between human and swine IAVs. No cases of human infection with swine strains were noticed, but direct infections of swine with H1N1pdm09 strains were detected. Moreover, we pointed out a continuous circulation of H1N1pdm09 strains in swine populations evidenced by the introduction of internal genes of this subtype. These events contribute to generating new viral variants—possibly endowed with pandemic potential—and emphasize the importance of continuous surveillance at both animal and human level.     

华南地区猪群猪流感病毒病原学和血清学调查

为了解华南地区猪群中猪流感病毒(SIV)的流行及其遗传变异情况,本研究从2016年~2017年广东、广西等地猪群236份猪肺脏病料组织和143份鼻拭子样品中分离鉴定得到3株SIV,全基因组测序和遗传演化分析结果显示,3个分离株均属于H1N1亚型欧亚类禽分支SIV,并且均与pdm09分支病毒株发生了重组。HA蛋白分子特征分析结果显示,A/Swine/Guangxi/NK/2016 HA蛋白第23位糖基化位点发生了缺失。3265份血清样品抗体监测结果显示,欧亚类禽H1N1、pdm09 H1N1和H3N2 SIV的血清抗体阳性率分别为27.53%、20.98%和34.85%。另外,0.64%的(21份)血清样品为H9N2亚型流感病毒抗体阳性,并且猪群中不同亚型和不同分支SIV之间混合感染的情况非常普遍。猪群中流感病毒血清抗体监测结果显示,EA H1N1、pdm09和H3N2亚型SIV HI抗体滴度最高均可达到1:1280,而H9N2亚型HI抗体滴度最高为1:160,表明H9N2 AIV虽然可以感染猪,但对猪还不适应。各月份的血清抗体阳性率分析显示,SIV的流行具有季节性,冬季(11月、12月和1月份)的流行最为严重。本研究可为华南地区猪群SI防控及疫苗株的筛选提供参考依据。     

Detection of Novel Reassortant Influenza A (H3N2) and H1N1 2009 Pandemic Viruses in Swine in Hanoi,Vietnam

From May to September 2013, monthly samples were collected from swine in a Vietnamese slaughterhouse for influenza virus isolation and serological testing. A(H1N1)pdm09 viruses and a novel H3N2 originating from reassortment between A(H1N1)pdm09 and novel viruses of the North American triple reassortant lineage were isolated. Serological results showed low seroprevalence for the novel H3N2 virus and higher seroprevalence for A(H1N1)pdm09 viruses. In addition, serology suggested that other swine influenza viruses are also circulating in Vietnamese swine.     

Syndromic survey and molecular analysis of influenza viruses at the human–swine interface in two West African cosmopolitan cities suggest the possibility of bidirectional interspecies transmission

Influenza viruses are frequently transmitted between pigs and their handlers, and among pig handlers. However, reports on socio‐environmental variables as potential risk factors associated with transmission of influenza in West African swine production facilities are very scarce. Syndromic survey for influenza was therefore conducted in Ibadan, Nigeria, and Kumasi, Ghana, in order to identify and elucidate selected socio‐environmental variables that may contribute to the occurrence and distribution of influenza‐like illness (ILI) among swine industry workers. In addition, molecular analyses were conducted to elucidate the nature of influenza viruses circulating at the human–swine interface in these cities and better understand the dynamics of their transmission. Influenza viruses were detected by type‐specific and subtype‐specific RT–PCR. Sequencing and phylogenetic analyses were carried out. Socio‐environmental variables were tested by both univariable and multivariable regression methods for significance at p < 0.05. Three risk factors for ILI were identified in each city. These included “frequency of visit of pig handler to pig pen or lairage” (Ibadan: risk ratio [RR] = 1.54, 95% confidence interval [CI] = 1.36–1.79, p = 0.02; Kumasi: RR = 1.28, 95% CI = 1.11–1.71, p = 0.01) and “pig handler's awareness about biosecurity measures” (Ibadan: RR = 7.09, 95% CI = 2.36–21.32, p < 0.001; Kumasi: RR = 4.84, 95% CI = 1.98–11.80, p < 0.001). Influenza A(H1N1)pdm09 viruses, with M genes closely related to those which circulated among pigs in the two cities during the same period, were detected among Nigerian and Ghanaian pig industry workers. These findings suggest the possibility of bidirectional transmission of influenza at the human–swine interface in these cities and underscore the need for more extensive molecular studies. Risk factors identified may assist in the control of human‐to‐human and human‐to‐swine transmission of influenza in the West African swine industry.     

Novel H1N2 swine influenza reassortant strain in pigs derived from the pandemic H1N1/2009 virus

Swine influenza monitoring programs have been in place in Italy since the 1990 s and from 2009 testing for the pandemic H1N1/2009 virus (H1N1pdm) was also performed on all the swine samples positive for type A influenza. This paper reports the isolation and genomic characterization of a novel H1N2 swine influenza reassortant strain from pigs in Italy that was derived from the H1N1pdm virus. In May 2010, mild respiratory symptoms were observed in around 10% of the pigs raised on a fattening farm in Italy. Lung homogenate taken from one pig showing respiratory distress was tested for influenza type A and H1N1pdm by two real time RT-PCR assays. Virus isolation was achieved by inoculation of lung homogenate into specific pathogen free chicken embryonated eggs (SPF CEE) and applied onto Caco-2 cells and then the complete genome sequencing and phylogenetic analysis was performed from the CEE isolate. The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT-PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double reassortant swine influenza viruses (SIVs), previously isolated in Sweden and Italy. NA sequences for these three strains were clustering with H3N2 SIVs. The emergence of a novel reassortant H1N2 strain derived from H1N1pdm in swine in Italy raises further concerns about whether these viruses will become established in pigs. The new reassortant not only represents a pandemic (zoonotic) threat but also has unknown livestock implications for the European swine industry.     

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.     

Serological Survey in Dogs and Cats for Influenza A(H1N1)pdm09 in Germany

A serological survey for the detection of antibodies to influenza A(H1N1)pdm09 was carried out in a population of dogs and cats in Germany. A total of 1150 sera collected in 2010 and 2011 were screened using an ELISA targeting anti‐nucleoprotein NP antibodies. Those initially screened positive samples were subsequently tested for antibodies to N1 neuraminidase followed by a virus neutralization test using A/Bayern/74/2009 strain. A prevalence of A(H1N1)pdm09‐specific antibodies of 0.13% and 1.93% was estimated among dogs and cats, respectively. Evidence of exposure to other influenza A virus subtypes was also observed.     

Surveillance of swine influenza viruses in sentinel familial farms in Hung Yen province in Northern Vietnam in 2013–2014

From May 2013 to April 2014, 15 swine family‐run farms (17 pig litters) in two districts in Hung Yen province, near Hanoi, were virologically and epizootiologically monitored for swine influenza viruses (SIV) monthly. No SIV was isolated from nasal swabs. Maternal antibodies were detected in 10 litters, and seroconversion against SIV was detected in six litters. There was a marked difference in patterns of SIV transmission in the two districts. Van Lam district which has low density of swine with mainly smallholder farms had low intensity of SIV, with much of the infection caused by H1N1 2009 pandemic‐like viruses A(H1N1)pdm09, likely originated from humans. In contrast, Van Giang district, which has high swine density and larger farms, had high levels of typical SIV (triple reassortants H3N2 and H3N2 Binh Duong lineage viruses) circulating within swine. With one exception, the SIV lineages detected were those we concurrently isolated from studies in a large central abattoir in Hanoi. Influenza‐like illness symptoms reported by farmers were poorly correlated with serological evidence of SIV infection.     

Serological surveillance and factors associated with influenza A virus in backyard pigs in Southern Brazil

Backyard pig populations are not monitored for influenza A virus (IAV) in Brazil and there are limited data about seroprevalence and risk factors in these populations. Our goal was to assess possible factors associated with IAV seroprevalence in backyard pig populations using an indirect ELISA protocol based on a recombinant nucleoprotein. Following the IAV screening using NP‐ELISA, subtype‐specific serology based on hemagglutination inhibition (HI) assay of the ELISA‐positive pigs was conducted. The survey comprised a total of 1,667 sera samples collected in 2012 and 2014 in 479 holdings and the estimated seroprevalence was 5.3% (3.84%–7.33%) and 2.3% (1.34%–3.71%) in the respective years. In both years, H1N1pdm09 was the most prevalent subtype. The multivariable analysis showed main factors such as “age,” “sex,” “number of suckling pigs” and “neighbours raising pigs” that presented the greatest effect on IAV seroprevalence in these pig populations. These factors may be associated with the low biosecurity measures and management of backyard holdings. In addition, the low IAV seroprevalences found in these backyard pig populations could be related to a low number of animals in each pig holding and low animal movement/replacement that do not favour IAV transmission dynamics. This low frequency of H1N1pdm09 seropositive pigs could also be due to sporadic human‐to‐pig transmission of what is now a human seasonal influenza A virus; however, these factors should be explored in future studies. Herein, these results highlight the importance of IAV continued surveillance in backyard pig holdings, since it is poorly known which IAVs are circulating in these populations and the risk they could pose to public health and virus transmission to commercial farms.     

Retracted: Risks of Avian Influenza (H5) in Duck Farms in the Ayeyarwaddy Delta Region,Myanmar

The Ayeyarwaddy delta region in the south‐west of Myanmar is the main agricultural and rice‐growing area. The region has a high density of duck and backyard chicken populations with low biosecurity. The objective of this study was to analyse risk factors for avian influenza (H5) in the Ayeyarwaddy delta region, Myanmar. A case–control risk factor study was conducted from April to June 2010 by individual interviews including risk factor questionnaires given to duck farmers (n = 50) in five townships in the Ayeyarwaddy delta region, Myanmar. Risk factor analyses were conducted using univariate analysis and multivariate logistic regression model with backward stepwise (wald) method. The results showed significant risk factors for AI (H5) sero‐positivity in ducks were wooden egg box containers (OR = 52.7, 95% CI = 2.34–1188, P = 0.013) and water sourced from wetlands (OR = 30.7, 95% CI = 1.96–481.6, P = 0.015). Conversely, the cleaning of reusable egg containers was determined as a protective factor (OR = 0.03, 95% CI = 0.00–0.42, P = 0.01). In conclusion, this study identified risk factors for AI (H5) in duck farms and the importance of avian influenza prevention and control.     

Comparison of embryonated chicken eggs with MDCK cell culture for the isolation of swine influenza virus

Embryonated chicken eggs (ECE) and the Madin-Darby canine kidney (MDCK) cell line were compared for isolation of swine influenza virus (SIV) from nasal swabs and tissue samples. Samples originated from 30 pigs experimentally inoculated with 2 × 10⁶ to 2 × 10⁷ embryo infectious dose 50% (EID₅₀)/mL of swine influenza strain A/Swine/Indiana/1726/88 (H1N1). The results were analyzed with McNemar's chi-squared test for symmetry. The results indicated that more samples were SIV-positive with ECE than with tissue culture (P ≤ 0.001), suggesting that ECE remains the system of choice for isolation of SIV. It is recommend that routine use of both SIV isolation systems will increase the sensitivity of detection of virus shedding by considering the differences in growth and tropism of diverse SIV strains.     

Descriptive clinical and epidemiological characteristics of influenza A H1N1 2009 virus infections in pigs in England

Infection of pigs with influenza A H1N1 2009 virus (A(H1N1)pdm09) was first detected in England in November 2009 following global spread of the virus in the human population. This paper describes clinical and epidemiological findings in the first English pig farms in which A(H1N1)pdm09 influenza virus was detected. These farms showed differences in disease presentation, spread and duration of infection. The factors likely to influence these features are described and relate to whether pigs were housed or outdoors, the age of the pigs, inter-current disease and the management system of the unit. Infection could be mild or clinically inapparent in breeding pigs with more typical respiratory disease being identified later in their progeny. Mortality was low where disease was uncomplicated by environmental stresses or concurrent infections. Where deaths occurred in pigs infected with A(H1N1)pdm09 influenza, they were mainly due to other infections, including streptococcal disease due to Streptococcus suis infection. This paper demonstrates the ease with which A(H1N1)pdm09 virus was transmitted horizontally and maintained in a pig population.     

Effects of freshwater crayfish on influenza A virus persistence in water

Several investigations have recently assessed the ability of some aquatic invertebrates to act as tools for avian influenza A virus (IAV) surveillance as well as their potential role(s) in IAV ecology. Because of this, as well as the high IAV seroprevalence rates noted in select mesocarnivores that commonly inhabit aquatic and semi‐aquatic habitats, we evaluated the effects that freshwater crayfish have on IAV in water at three dose levels and monitored for the presence of IAV in crayfish tissues (gill and green gland) and haemolymph at multiple time points. At relatively high, medium and low (approximately 10⁴, 10³ and 10² EID₅₀/ml, respectively) doses, mesocosms containing crayfish (Orconectes sp.) had less detectable IAV RNA present when final water samples were assayed (9 days post‐contact [DPC]). In general, containers without crayfish present had nearly three‐fold greater quantities of viral RNA at 9 DPC. A varying number of RNA positive samples were detected for the three crayfish sample types collected. Gill tissue produced the largest number of positive non‐water samples (n = 26), with the highest quantities detected from crayfish sampled on 1 and 4 DPC (10^3.5 EID₅₀ equivalent/ml). On a few occasions, gill (n = 8) and haemolymph samples (n = 1) produced higher quantities of viral RNA than their respective water samples or water samples collected 1–2 DPC earlier, but these differences were typically minor. Based upon water samples, statistical models indicated that the interaction of dose and crayfish exposure days explained most of the variation in these data. Future efforts should address if crayfish exposed to IAV‐laden water have the capacity to successfully transmit IAVs to mammals and birds which frequently prey upon them.     

Serological and virological surveillance of swine H1N1 and H3N2 influenza virus infection in two farms located in Hubei province, central China

Swine influenza viruses H1N1 and H3N2 have been reported in the swine population worldwide. From June 2008 to June 2009, we carried out serological and virological surveillance of swine influenza in the Hubei province in central China. The serological results indicated that antibodies to H1N1 swine influenza virus in the swine population were high with a 42.5% (204/480) positive rate, whereas antibodies to H3N2 swine influenza virus were low with a 7.9% (38/480) positive rate. Virological surveillance showed that only one sample from weanling pigs was positive by RT-PCR. Phylogenetic analysis of the hemagglutinin and neuraminidase genes revealed that the A/Sw/HB/S1/2009 isolate was closely related to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses. In conclusion, H1N1 influenza viruses were more dominant in the pig population than H3N2 influenza viruses in central China, and infection with avian-like H1N1 viruses persistently emerged in the swine population in the area.     

Hepatitis E virus infection in swine workers: A meta‐analysis

Hepatitis E virus (HEV) infects both humans and animals. Swine has been confirmed to be the principal natural reservoir, which raises a concern that HEV infection would be substantially increasing among swine workers. The present study calculated the pooled prevalence of IgG antibodies against HEV among swine workers and the general population in previous cross‐sectional studies. We conducted a meta‐analysis comparing the prevalence of HEV infection between swine workers and the general population, including local residents, blood donors and non‐swine workers. Through searches in three databases (PubMed and OVID in English, and CNKI in Chinese) and after study selection, a total of 32 studies from 16 countries (from 1999 through 2018) were included in the meta‐analysis. A random‐effect model was employed in the study; an I ² statistic assessed heterogeneity, and the Egger's test detected publication bias. The comparative prevalence of anti‐HEV IgG was pooled from the studies. Compared to the general population, the prevalence ratio (PR) for swine workers was estimated to be 1.52 (95% CI 1.38–1.76) with the I ² being 71%. No publication bias was detected (p = 0.40). A subgroup analysis further indicated increased prevalence of anti‐HEV IgG in the swine workers in Asia (PR = 1.49, 95% CI: 1.35–1.64), in Europe (PR = 1.93, 95% CI: 1.49–2.50) and in all five swine‐related occupations, including swine farmers, butchers, meat processors, pork retailers and veterinarians (PR ranged between 1.19 and 1.75). In summary, swine workers have a relatively higher prevalence of past HEV infection, and this finding is true across swine‐related occupations, which confirms zoonotic transmission between swine and swine workers.     

H3N2亚型猪流感病毒HA基因序列测定及抗原性分析

采用RT-PCR技术对4株H3N2亚型猪流感病毒的HA基因进行了扩增,将获得的PCR产物分别与pMD18-T克隆载体连接,进行序列测定。测序结果显示,4个毒株均含有完整的开放阅读框,并且均未发现核苷酸插入或缺失现象;分离毒株间核苷酸同源性为99.4%~99.7%,氨基酸同源性为98.2%~99.3%。同源性分析表明,4个毒株与2003年的猪流感病毒广东分离株有很高同源性(均在99%以上),说明近段时间我国H3N2亚型的猪流感病毒变异不大,重组的频率不是很高,同时又与人流感病毒香港分离株有较高的同源性(均为99.4%)。交叉血凝抑制试验显示,S3株与其他3毒株抗原性差异明显。鉴于猪在流感病毒传播与复制间的特殊地位,应密切监测猪流感。     

猪流感与公共卫生

猪流感(Swine influenza,SI)是目前危害全世界养猪业的重要呼吸道传染病之一.导致猪发病的致病毒株主要有H1N1、H1N2、H3N1、H3N2、H2N3、H5N1和H9N2等亚型流感病毒,特别是从猪体分离H5N1和H9N2亚型流感病毒对禽流感的控制及人类公共卫生有重要意义.针对目前流行的甲型H1N1疫情,对猪流感病毒的分子生物学、临床症状、病理变化及公共卫生意义等方面进行了综述,以期对其有一个较为全面的了解.