Use of a Multiplex RT-PCR Assay for Simultaneous Detection of the North American Genotype Porcine Reproductive and Respiratory Syndrome Virus,Swine Influenza Virus and Japanese Encephalitis Virus

A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Specific primers for each of the 3 RNA viruses,North American genotype porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and swine influenza virus,were used in the testing procedure.The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses.The assay was also effective in detecting one or more of the same viruses in various combinations in specimens,including lymph nodes,lungs,spleens,and tonsils,collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses.The results from the multiplex RT-PCR were confirmed by virus isolation.The relative efficiency(compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.

Porcine Interleukin-2 Expression in Insect Cells and Its Enhancement of Pig Immunity to Swine Influenza Virus Inactivated Vaccine

Mature porcine interleukin-2 （pIL-2） gene was amplified by PCR from the plasmid pGEM-T-pIL2 and cloned into the baculovirus pFastBacTM Dual vector of the Bac-to-Bac baculovirus expression system under the control of the PH promoter. Recombinant plL-2 （rpIL-2） expressed in Sf9 insect cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunofluorescence assay. Western blot analysis confirmed that the rpIL-2 protein had a molecular mass of 20 kDa, which was larger than the molecular mass of the mature protein predicted based on its peptide sequence. The rpIL-2 protein induced in vitro proliferation of ConA-stimulated porcine splenocytes and enhanced in vivo protective immune responses induced by vaccinating the pigs with inactivated oil emulsion vaccine against swine influenza virus. The results showed that the rpIL-2 expressed in Sf9 insect cells has immunoenhancement effects; the finding lays the foundation for the preparation of a specific recombinant IL-2 protein and the development of a novel immune adjuvant of vaccines against various infectious porcine pathogens to increase the immunoprotective efficacy of vaccines.

Diagnosis and Prevention of Swine Influenza

Swine influenza （SI） is an acute, febrile, highly contagious, respiratory infectious disease caused by swine influenza virus （SIV）, which is a common disease in intensive pig farms, and prevalent throughout the world. Disease in affected pigs was characterized by burst, high fever, rapid epidemic spread. The mortality of SI with secondary or mixed infection could be increased. Moreover, more researches showed that the outbreaks of human influenza are associated with the outbreak of swine influenza, and there are a striking parallel nature and relevance. In the present article, the pathogen, epidemiology, diagnosis, treatment and public health aspects were elaborated, so as to provide a reference for scientific prevention and treatment of SI.

Association of swine influenza H1N1 pandemic virus(SIV-H1N1p) with porcine respiratory disease complex in sows from commercial pig farms in Colombia

Porcine respiratory disease complex （PRDC） is a serious health problem that mainly affects growing and finishing pigs. PRDC is caused by a combination of viral and bacterial agents, such as porcine reproductive and respiratory syndrome virus （PRRSV）, swine influenza virus （SIV）, Mycoplasma hyopneumoniae （Myh）, Actinobacillus pleuropneumoniae （APP）, Pasteurella multocida and Porcine circovirus 2 （PCV2）. To characterize the specific role of swine influenza virus in PRDC presentation in Colombia, 11 farms from three major production regions in Colombia were examined in this study. Nasal swabs, bronchial lavage and lung tissue samples were obtained from animals displaying symptoms compatible with SIV. Isolation of SIV was performed in 9-day embryonated chicken eggs or Madin-Darby Canine Kidney （MDCK） cells. Positive isolates, identified via the hemagglutination inhibition test, were further analyzed using PCR. Overall, 7 of the 11 farms were positive for SIV. Notably, sequencing of the gene encoding the hemagglutinin （HA） protein led to grouping of strains into circulating viruses identified during the human outbreak of 2009, classified as pandemic H1N1-2009. Serum samples from 198 gilts and multiparous sows between 2008 and 2009 were obtained to determine antibody presence of APP, Myh, PCV2 and PRRSV in both SIV-H1Nlp-negative and -positive farms, but higher levels were recorded for SIV- HI Nlp-positive farms. Odds ratio （OR） and P values revealed statistically significant differences （p〈0.05） in PRDC presentation in gilts and multiparous sows of farms positive for SIV-HINlp. Our findings indicate that positive farms have increased risk of PRDC presentation, in particular, PCV2, APP and Myh.

Nested RT-PCR method for the detection of European avian-like H1 swine influenza A virus

Swine influenza A virus（swine IAV） circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like（EA） H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription（RT）-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets（outer and inner） were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit（PFU） m L^（-1） which was over 10~4 PFU m L^（-1） for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.

Construction and Immunogenicity of a Recombinant Adenovirus Expressing the HA Gene of H5N1 Subtype Swine Influenza Virus

To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID50 of the rAd-H5HA- EGFP was assessed to be 2.26×1010/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.

Identification and Treatment of Swine Influenza and Common Cold in Veterinary Clinic

[Objective] The paper was to investigate the differentiation and treatment of swine influenza and common cold in veterinary clinic.[Method] The clinical data of diseased pigs from May 2017 to May 2018 were analyzed retrospectively to explore the clinical treatment methods for swine influenza and common cold, and the effectiveness and accuracy of clinical diagnosis was further improved. [Result] Through in-depth analysis of test results, we obtained a comprehensive understanding of the main causes of swine influenza and common cold, and found that the causes in-cluded improper breeding management of pigs, nutrition of pigs and lack of immunity of newborn pigs. [Conclusion] Analysis of the causes of swine influenza and common cold and suggestion of targeted clinical treatment measures will reduce the incidence of swine disease, and promote the vet-erinary clinical treatment effect of swine influenza and common cold.

Cloning, Prokaryotic Expression, and Antigenicity Analysis of NS1 Gene of H9N2 Swine Influenza Virus

To obtain the NS1 gene of swine influenza virus H9N2 subtype expressed efficiently in E. coli, to develope the effective diagnostic methods for swine influenza virus H9N2 subtype, the NS 1 gene of swine influenza virus H9N2 subtype was amplified by reverse transcriptase polymerase chain reaction （RT-PCR） and cloned into a prokaryotic expression vector, pET-28a（＋）, and overexpressed in E. coli BL21-DE3 after induction with 5 mmol L-1 lactose. The recombinant protein was purified by Ni-NTA and identified by western-blotting. An indirect enzyme-linked immunosorbent assay （ELISA） was used to analyze the antigenicity of the recombinant protein. The recombinant protein of NS1 was about 26 kD. The Western-blotting test showed that the recombinant protein reacted specifically with positive sera. The results of the ELISA test showed that the recombinant protein had good antigenicity.

The epidemiology of swine influenza

Globally swine influenza is one of the most important diseases of the pig industry, with various subtypes of swine influenza virus co-circulating in the field. Swine influenza can not only cause large economic losses for the pig industry but can also lead to epidemics or pandemics in the human population. We provide an overview of the pathogenic characteristics of the disease, diagnosis, risk factors for the occurrence on pig farms, impact on pigs and humans and methods to control it. This review is designed to promote understanding of the epidemiology of swine influenza which will benefit the control of the disease in both pigs and humans.

First isolation and identification of H1N1 swine influenza viruses in Colombian pig farms

The pig industry in Colombia has grown 30% in the last decade achieving high levels of technology and efficiency;in spite of that, respiratory diseases remain a constraint. Since 1970, serological evidence and histological findings suggested the role of swine influenza virus (SIV) as part of the porcine respiratory disease complex;nevertheless, elusive and molecular typing isolates are missing. This study was aimed at isolating SIV from intensive pig farms and to achieve molecular characterization to determine strains circulating in the field. In order to accomplish this goal, 242 samples were taken from nasal swabs, 25 from bronchial washes and 8 from lung tissue. Samples were collected during a period of three years, between 2008 and 2010 and were originated from 78 farms of the three main pig production regions of the country. The samples were transported in BHI broth with 2% antibiotic and antimycotic solution and stored at –70?C until processed. The swabs were inoculated in 9 - 11 days old embryo chicken eggs and in MDCK (Madin Darby Canine Kidney) cell cultures with the addition of trypsin. The isolates were identified by the HA (hemoagglutination) test and by RT-PCR targeting the HA (hemagglutinin), NA (Neuraminidase) and M (Matrix) genes. Full length sequence of the HA and NA glycoproteins from four selected virus isolates was conducted (Macrogen?. USA). As a result, fifteen SIV isolates from nine farms distributed in the three regions were obtained. Twelve of the isolates are related to the swine origin H1N1 virus that caused the 2009 influenza pandemic. The remaining three viruses were related to classical swine influenza viruses.

Establishing an ELISA,Based on the HA1 Protein,for Detecting Antibodies against H3N2 Subtype Swine Influenza Virus

The HA1 gene of H3N2 subtype swine influenza virus(SIV)was cloned into the expression plasmid pET-30a,the recombinant plasmid was named pET-HAl.This was transformed into E.coli BL21(DE3),and expressed by induction with IPTG.The expressed HA protein was identified by SDS-PAGE and Western blotting which showed the protein to be 42kDa and was immunoreactive.The purified HA protein was used to establish the indirect ELIS A for detection of the antibodies,specifically against the H3 subtype of SIV.The assay has excellent specificity,sensitivity and reproducibility.When 96 serum samples,randomly collected from the field,were evaluated in parallel by this new ELISA using recombinant HA1 and a routine HI test,the coincidental rate between the two tests was 86.5%.These results show that the recombinant HAl-based ELISA is specific,sensitive and easy to perform for the serological diagnosis of SIV infection.

The evolution,pathogenicity and transmissibility of quadruple reassortant H1N2 swine influenza virus in China:A potential threat to public health

Swine are regarded as“intermediate hosts”or“mixing vessels”of influenza viruses,capable of generating strains with pandemic potential.From 2020 to 2021,we conducted surveillance on swine H1N2 influenza(swH1N2)viruses in swine farms located in Guangdong,Yunnan,and Guizhou provinces in southern China,as well as Henan and Shandong provinces in northern China.We systematically analyzed the evolution and pathogenicity of swH1N2 isolates,and characterized their replication and transmission abilities.The isolated viruses are quadruple reassortant H1N2 viruses containing genes from pdm/09 H1N1(PB2,PB1,PA and NP genes),triple-reassortant swine(NS gene),Eurasian Avian-like(HA and M genes),and recent human H3N2(NA gene)lineages.The NA,PB2,and NP of SW/188/20 and SW/198/20 show high gene similarities to A/Guangdong/Yue Fang277/2017(H3N2).The HA gene of swH1N2 exhibits a high evolutionary rate.The five swH1N2 isolates replicate efficiently in human,canine,and swine cells,as well as in the turbinate,trachea,and lungs of mice.A/swine/Shandong/198/2020 strain efficiently replicates in the respiratory tract of pigs and effectively transmitted among them.Collectively,these current swH1N2 viruses possess zoonotic potential,highlighting the need for strengthened surveillance of swH1N2 viruses.

Epidemiology and Genotypic Diversity of Eurasian Avian-Like H1N1 Swine Influenza Viruses in China

Eurasian avian-like H1 N1(EA H1 N1)swine influenza virus(SIV)outside European countries was first detected in Hong Kong Special Administrative Region(Hong Kong,SAR)of China in 2001.Afterwards,EA H1 N1 SIVs have become predominant in pig population in this country.However,the epidemiology and genotypic diversity of EA H1 N1 SIVs in China are still unknown.Here,we collected the EA H1 N1 SIVs sequences from China between 2001 and 2018 and analyzed the epidemic and phylogenic features,and key molecular markers of these EA H1 N1 SIVs.Our results showed that EA H1 N1 SIVs distributed in nineteen provinces/municipalities of China.After a long-time evolution and transmission,EA H1 N1 SIVs were continuously reassorted with other co-circulated influenza viruses,including 2009 pandemic H1 N1(A(H1 N1)pdm09),and triple reassortment H1 N2(TR H1 N2)influenza viruses,generated 11 genotypes.Genotype 3 and 5,both of which were the reassortments among EA H1 N1,A(H1 N1)pdm09 and TR H1 N2 viruses with different origins of M genes,have become predominant in pig population.Furthermore,key molecular signatures were identified in EA H1 N1 SIVs.Our study has drawn a genotypic diversity image of EA H1 N1 viruses,and could help to evaluate the potential risk of EA H1 N1 for pandemic preparedness and response.

Swine-origin influenza-virus-induced acute lung injury:Novel or classical pathogenesis?

Influenza viruses are common respiratory pathogens in humans and can cause serious infection that leads to the development of pneumonia.Due to their hostrange diversity,genetic and antigenic diversity,and potential to reassort genetically in vivo,influenza A viruses are continual sources of novel influenza strains that lead to the emergence of periodic epidemics and outbreaks in humans.Thus,newly emerging viral diseases are always major threats to public health.In March 2009,a novel influenza virus suddenly emerged and caused a worldwide pandemic.The novel pandemic influenza virus was genetically and antigenically distinct from previous seasonal human influenza A/H1N1 viruses;it was identified to have originated from pigs,and further genetic analysis revealed it as a subtype of A/H1N1,thus later called a swine-origin influenza virus A/H1N1.Since the novel virus emerged,epidemiological surveys and research on experimental animal models have been conducted,and characteristics of the novel influenza virus have been determined but the exact mechanisms of pulmonary pathogenesis remain to be elucidated.In this editorial,we summa-rize and discuss the recent pandemic caused by the novel swine-origin influenza virus A/H1N1 with a focus on the mechanism of pathogenesis to obtain an insight into potential therapeutic strategies.

猪流感病毒的流行现状及疫苗研究进展

猪流感(swine influenza,SI)是猪的重要呼吸道疾病之一,其特征为发热、咳嗽、呼吸困难、食欲不振等。临床上SI感染病死率较低,但常导致饲料利用率下降,生长性能差,可与其他病原体合并感染而引起高发病率甚至死亡。猪在流感种间传播中起着复杂且重要的作用,SI的发生不仅给养猪业造成巨大经济损失,对公共卫生也造成严重威胁。论文对近10年我国猪流感病毒(Swine influenza virus,SIV)感染的流行病学、病毒进化、跨物种传播、疫苗研发等情况进行综述,分析了综合防控策略,为SI的风险评估和综合防控提供参考。

一株猪流感病毒A/swine/Shanghai/01/2019(H1N1)的分离鉴定及遗传进化分析

为鉴定上海地区猪流感病毒的流行株及其分子生物学特征,本研究从上海地区养殖场采集疑似流感症状的猪咽拭子样品40份,采用套式PCR结合鸡胚分离鉴定方法,从中分离到1株H1N1亚型猪流感病毒(SIV),命名为A/swine/Shanghai/01/2019(H1N1)。经全基因组测定和遗传进化分析结果显示该分离株的8个基因节段与欧亚类禽H1N1 SIV同源性最高,其PB2、PB1、PA基因节段可能来源于A/swine/Jiangsu/49/2012(H1N1)病毒,NA、NP和M基因节段可能来源于A/swine/Shanghai/3/2014(H1N1)病毒。该分离病毒的HA蛋白裂解位点为PSIQSR↓GLFGAI,为低致病性流感病毒的分子特征。该分离株NA蛋白未出现与神经氨酸酶抑制剂相关的氨基酸突变,M2蛋白S31N突变表明该病毒可能对金刚烷胺类药物具有一定的耐药性。PA蛋白224S和PB2蛋白701N突变提示该分离株对哺乳动物的适应性增强,可能导致其致病性增强,需要加强对此类SIV的监测。结合近年来的监测表明,上海地区猪群中H1N1亚型SIV持续存在,需要持续跟踪监测SIV变异趋势。本研究为防控猪流感提供实验依据。

猪流感病毒分离株A/Swine/Fujian/F1/2001(H5N1)表面蛋白基因序列分析

对猪流感病毒株A/Swine/Fujian/F1/2001(H5N1)(简称SW/FJ/F1/01)的表面蛋白基因进行序列测定,并与GenBank中的A型流感病毒H5N1亚型毒株参考序列进行比较。遗传进化分析表明,猪流感病毒SW/FJ/F1/01的血凝素基因(HA)和神经氨酸酶基因(NA)均与2000年浙江鸭源毒株A/Duck/Zhejiang/52/2000(H5N1)(简称DK/ZJ/52/00)的亲缘关系最近,其HA蛋白的裂解位点为-RRKKR*G-,受体结合位点226位为Q,228位为G,识别唾液酸-α2,3-半乳糖,与人流感病毒H3亚型的受体结合位点不同,分析表明该病毒来源于禽源流感病毒。

H9N2亚型猪流感病毒A/Swine/Shandong/1/02分离株的基因序列分析

从有肺炎症状的病猪中分离到1株H9N2亚型猪流感病毒（SIV）A／Swine／Shandong／1／02，对其进行了全病毒基因序列分析。结果表明，该毒株8个基因片段的核苷酸序列均来自禽流感病毒（AIV），与我国目前家禽中流行的H9N2亚型AIV毒株具有很高的同源性，与A／Duck／Hong Kong／Y280／97（H9N2）的同源性为94．1％～98．9％，与A／Chicken／Beijing／1／94（H9N2）的同源性为94．5％～98．2％；推导的其血凝素（HA）裂解位点处的氨基酸序列为P—A-R—S-S-R，完全符合H9亚型AIV欧亚分支中的类A／Chicken／Beijing／1／94亚分支的特征；基因分析结果表明，该分离株的所有基因片段均来源于H9N2亚型AIV，它可直接感染猪，并导致发病，但它并未在猪体内发生重组。

人源H3N2亚型猪流感病毒A/Swine/Guangdong/1/2005的分离鉴定及全基因遗传演化分析

2005年从广东某猪场采集疑似流感病猪的病料(气管和肺脏)样品共40份,通过MDCK细胞培养分离到6株A型流感病毒,经过血凝抑制和PCR扩增方法鉴定均为H3N2亚型。挑选其中一个代表株A/swine/Guangdong/1/05(H3N2)(SwGD1/05)进行全基因组测序并与GenBank中相关序列比较,结果发现SwGD1/05的8个基因分别与人流感病毒相关基因氨基酸同源性高达98.5%～99.4%;SwGD1/05的8个基因进化树中分别位于北美和欧洲人流感病毒进化谱系位置;由此推测SwGD1/05可能来源于北美或欧洲人源流感病毒株。这一结果为阐明猪作为流感病毒传播的中间宿主作用提供了的理论支持,本研究具有重要的人类公共卫生意义。

猪流感病毒A/Swine/Inner Mongolia/547/01分离株神经氨酸酶基因的克隆及真核表达

根据GenBank中登录的猪流感病毒参照序列设计了扩增神经氨酸酶 (NA)基因的特异性引物。从猪流感病毒A/Swine/InnerMongolia/5 4 7/0 1(H3N2 )株感染的鸡胚液中直接提取病毒RNA ,经RT_PCR扩增后将其克隆到pMD18_T载体上 ,进行序列测定和拼接。用HindⅢ酶切后 ,亚克隆到杆状病毒转移载体pMelBacB上 ,得到重组转移载体pMelBacNA ,然后与线性化杆状病毒DNA(Bac_N_BlueTMDNA)共转染于sf9昆虫细胞。经 3轮噬斑纯化 ,提取其DNA经PCR鉴定 ,获得重组杆状病毒株。SDS_PAGE结果显示 ,目的基因已获得表达 ,Westernblot和神经氨酸酶活性检测结果显示表达产物具有良好的免疫原性和神经氨酸酶活性 。