Isolation of a porcine respiratory,non‐enteric coronavirus related to transmissible gastroenteritis

Summary

A porcine respiratory, non‐enteric virus which is related to the coronavirus transmissible gastroenteritus virus (TGEV) has been isolated in pigs and in cell culture. The isolate was designated TLM 83.

It has become very widespread and enzootic among the swine population in Belgium and in other swine raising countries. It causes an infection of the lungs and appears to spread by aerogenic route. It does not replicate in the enteric tract. The experimental infection in conventional and gnotobiotic pigs in isolation remains subclinical. The infection, either experimental or in the field, results in the formation of antibodies which neutralise the classical enteric TGEV. Based on this relationship, this virus is assumed to be a new TGEV‐related porcine respiratory coronavirus or TGEV itself which has totally lost its tropism for the enteric tract.     

Differentiation between transmissible gastroenteritis virus and porcine respiratory coronavirus using a cDNA probe.

A plasmid, pG3BS, containing a cDNA clone from the 5' coding region of the peplomer glycoprotein gene appears to be specific for enteric transmissible gastroenteritis virus (TGEV) strains and for live-attenuated TGEV vaccines. This cDNA probe is used to differentiate porcine respiratory coronavirus (PRCV) isolates from TGEV field and vaccine strains by a slot blot hybridization assay. Probe pG3BS also hybridizes to canine coronavirus (CCV) RNA but does not hybridize to antigenically related feline infectious peritonitis virus (FIPV) RNA. The RNAs of 13 enteric TGEV isolates from the United States, Japan, and England, 4 US-licensed live-attenuated TGEV vaccines, and antigenically closely related CCV were detected by pG3BS. The RNAs of FIPV and 3 US isolates of PRCV did not react with pG3BS but were detected by a TGEV-derived plasmid, pRP3. Pigs infected with either PRCV or TGEV test serologically positive for TGEV antibody by the serum neutralization test. Characterization of the virus circulating in a swine herd by the pG3BS probe will differentiate between an enteric TGEV and a respiratory PRCV infection.     

Infection with porcine respiratory coronavirus does not fully protect pigs against intestinal transmissible gastroenteritis virus

Eight nine-week-old specific-pathogen-free pigs which had been infected with the transmissible gastroenteritis virus (TGEV)-related porcine respiratory coronavirus (PRCV) and four uninfected littermates were challenged with TGEV. The previous PRCV infection failed to protect them against the enteric TGEV infection. Virus excretion in faeces was detected by an ELISA in all the pigs for three to six consecutive days after inoculation. Although little diarrhoea was observed, the infection extended through much of the small intestine of one of the previously infected pigs four days after inoculation. Challenge with TGEV caused a secondary neutralising antibody response. By using a peroxidase conjugate of a monoclonal antibody which recognises a specific antigenic site on TGEV, antibodies against TGEV could be distinguished from antibodies against PRCV in an ELISA blocking test.     

Evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the United States

A respiratory variant of transmissible gastroenteritis virus (TGEV), designated PRCV-Ind/89, was isolated from a swine breeding stock herd in Indiana. The virus was readily isolated from nasal swabs of pigs of different ages and induced cytopathology on primary porcine kidney cells and and on a swine testicular (ST) cell line. An 8-week-old pig infected oral/nasally with the respiratory variant and a contact pig showed no signs of respiratory or enteric disease. These pigs did not shed virus in feces but did shed the agent from the upper respiratory tract for approximately 2 weeks. Baby pigs from 2 separate litters (2 and 3 days old) also showed no clinical signs following oral/nasal inoculation with PRCV-Ind/89. In a third litter, 5 of 7 piglets (5 days old) infected either oral/nasally or by stomach tube developed a transient mild diarrhea with villous atrophy. However, virus was not isolated from rectal swabs or ileal homogenates of these piglets, and viral antigen was not detected in the ileum by fluorescent antibody staining even though the virus was easily recovered from nasal swabs and lung tissue homogenates. Swine antisera produced against PRCV-Ind/89 or enteric TGEV cross-neutralized either virus. In addition, an anti-peplomer monoclonal antibody, 4F6, that neutralizes TGEV also neutralized the PRCV-Ind/89 isolate. Radioimmunoassays with a panel of monoclonal antibodies indicated that the Indiana respiratory variant and the European PRCV are antigenically similar.     

Field validation of a commercial blocking ELISA to differentiate antibody to transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus and to identify TGEV-infected swine herds.

A commercially available blocking ELISA was analyzed for its ability to identify antibodies to porcine coronaviruses (transmissible gastroenteritis virus [TGEV] or porcine respiratory coronavirus [PRCV]), to differentiate antibodies to TGEV and PRCV, and to identify TGEV-infected herds. Nine sera from uninfected pigs, 34 sera from 16 pigs experimentally infected with TGEV, and sera from 10 pigs experimentally infected with PRCV were evaluated using both the TGEV/PRCV blocking ELISA and a virus neutralization (VN) assay. The ELISA was not consistently effective in identifying pigs experimentally infected with TGEV until 21 days postinfection. Sera from 100 commercial swine herds (1,783 sera; median 15 per herd) were similarly evaluated using both tests. Thirty of these commercial herds had a clinical history of TGEV infection and a positive TGEV fluorescent antibody test recorded at necropsy within the last 35 months, while 70 herds had no history of clinical TGEV infection. The blocking ELISA and the VN showed good agreement (kappa 0.84) for the detection of porcine coronavirus antibody (TGEV or PRCV). The sensitivity (0.933) of the ELISA to identify TGEV-infected herds was good when considered on a herd basis. The ELISA was also highly specific (0.943) for the detection of TGEV-infected herds when the test results were evaluated on a herd basis. When sera from specific age groups were compared, the ELISA identified a greater proportion (0.83) of pigs in herds with TGEV antibody when suckling piglets were used. In repeatability experiments, the ELISA gave consistent results when the same sera were evaluated on different days (kappa 0.889) and when sera were evaluated before and after heating (kappa 0.888). The blocking ELISA was determined to be useful for herd monitoring programs and could be used alone without parallel use of the VN assay for the assessment of large swine populations for the detection of TGEV-infected herds.     

Transmissible gastroenteritis virus infection: a vanishing specter

About twenty years ago, a new coronavirus, porcine respiratory coronavirus (PRCoV), was detected in swine herds. This virus is related to transmissible gastroenteritis virus (TGEV); however, it is not enteropathogenic but causes only minor respiratory symptoms. As PRCoV shares some epitopes for neutralizing antibodies with TGEV, it acts like a nature-made vaccine against TGEV resulting in a drastic reduction of TGE outbreaks in Europe. A major difference between the two porcine coronaviruses is a large deletion in the surface protein S gene of PRCoV. Because of this structural difference, TGEV but not PRCoV has a sialic acid binding activity that allows the attachment to mucins and mucin-type glycoproteins. The sialic acid binding activity may allow TGEV to overcome the mucus barrier in the gut and to get access to the intestinal epithelium for initiation of infection.     

Efficacy of antiserum produced in goats and pigs to passively protect piglets against virulent transmissible gastroenteritis virus.

The protective effect of sera produced in swine and goats exposed to virulent transmissible gastroenteritis virus (TGEV) or modified-live TGEV was tested in hysterectomy-derived, colostrum-deprived three-day-old pigs. Pigs were given serum with their daily ration of milk, and their immunity to virulent TGEV was determined. The pigs were observed for ten days for clinical signs of TGEV infection. One of nine pigs receiving goat serum was protected whereas all three pigs receiving three doses of swine serum per day were protected. Because virus was not isolated from the goats after oral/intranasal vaccination, it is suggested the virus did not replicate in either the respiratory or digestive tract of the goat.     

Detection of respiratory pathogens in air samples from acutely infected pigs

Pathogens causing significant respiratory disease in growing pigs include Porcine reproductive and respiratory syndrome virus, Porcine circovirus 2, swine influenza virus, porcine respiratory coronavirus, Mycoplasma hyopneumoniae, and Bordetella bronchiseptica. The objective of this research was to characterize the respiratory excretion of these pathogens by acutely infected pigs. Pigs were inoculated under experimental conditions with 1 pathogen. Samples were collected from the upper respiratory tract and exhaled air. All pathogens were detected in swabs of the upper respiratory tract, but only M. hyopneumoniae and B. bronchiseptica were detected in expired air from individually sampled, acutely infected pigs. These findings suggest either that the acutely infected pigs did not aerosolize the viruses or that the quantity of virus excreted was below the detection threshold of current sampling or assay systems, or both, at the individual-pig level.     

A competitive inhibition ELISA for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (TGEV) or with the TGEV-related porcine respiratory coronavirus

A competitive inhibition ELISA was developed to detect non-neutralizing antibodies to the peplomer protein of transmissible gastroenteritis virus (TGEV) in porcine sera using a monoclonal antibody as an indicator. It was demonstrated that field strains of the TGEV-related porcine respiratory coronavirus (PRCV) did not induce this antibody, whereas the Miller strain and field strains of TGEV did. The sensitivity of the competitive inhibition ELISA appeared to be similar to that of the virus neutralization (VN) test. The test enables differentiation of pigs which were previously infected with TGEV or PRCV and which cannot be distinguished by the classical anti-TGEV neutralization test. The present test is useful for selective serodiagnosis.     

Swine coronaviruses (SCoVs) and their emerging threats to swine population,inter-species transmission,exploring the susceptibility of pigs for SARS-CoV-2 and zoonotic concerns

Swine coronaviruses (SCoVs) are one of the most devastating pathogens affecting the livelihoods of farmers and swine industry across the world. These include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine delta coronavirus (PDCoV). Coronaviruses infect a wide variety of animal species and humans because these are having single stranded-RNA that accounts for high mutation rates and thus could break the species barrier. The gastrointestinal, cardiovascular, and nervous systems are the primary organ systems affected by SCoVs. Infection is very common in piglets compared to adult swine causing high mortality in the former. Bat is implicated to be the origin of all CoVs affecting animals and humans. Since pig is the only domestic animal in which CoVs cause a wide range of diseases; new coronaviruses with high zoonotic potential could likely emerge in the future as observed in the past. The recently emerged severe acute respiratory syndrome coronavirus virus-2 (SARS-CoV-2), causing COVID-19 pandemic in humans, has been implicated to have animal origin, also reported from few animal species, though its zoonotic concerns are still under investigation. This review discusses SCoVs and their epidemiology, virology, evolution, pathology, wildlife reservoirs, interspecies transmission, spill-over events and highlighting their emerging threats to swine population. The role of pigs amid ongoing SARS-CoV-2 pandemic will also be discussed. A thorough investigation should be conducted to rule out zoonotic potential of SCoVs and to design appropriate strategies for their prevention and control.     

Seroprevalence of economically important viral pathogens in swine populations of Trinidad and Tobago,West Indies

The objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (SwIV), as well as the seroprevalence of porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) in pigs in Trinidad and Tobago (T&T). Blood samples (309) were randomly collected from pigs at farms throughout T&T. Serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial ELISA kits, and the circulating strains of SwIV were identified by the hemagglutination inhibition test (HIT). Antibodies against SwIV were detected in 114 out of the 309 samples (37%). Out of a total of 26 farms, 14 tested positive for SwIV antibodies. HI testing revealed high titers against the A/sw/Minnesota/593/99 H3N2 strain and the pH1N1 2009 pandemic strain. Antibodies against PPV were detected in 87 out of the 309 samples (28%), with 11 out of 26 farms testing positive for PPV antibodies. Antibodies against PCV-2 were detected in 205 out of the 309 samples tested (66%), with 25 out of the 26 farms testing positive for PCV-2 antibodies. No antibodies were detected in any of the tested pigs to PRRSV, TGEV, PRCV, or CSFV.     

Production of porcine aminopeptidase N (pAPN) site‐specific edited pigs

Porcine viral diarrhea is an acute and highly contagious enteric disease in pigs which causes huge economic losses in pig industry worldwide. Transmissible gastroenteritis virus (TGEV) is main pathogens responsible for piglets viral diarrhea. Knockout the host cellular surface receptor for TGEV may be an effective way to accelerate the breeding of resistant pigs. In this study, we applied site‐specific editing pAPN which is effective in swine testis (ST) cells. Site‐specific editing of pAPN reduced TGEV proliferation in ST cells by 96%–99% at different time periods post‐infection. Next, the site‐specific editing of pAPN porcine fetal fibroblasts were produced, and then the cell colonies were used as donor cells to generate the site‐specific editing of pAPN pigs. Our research findings will not only offer a more thorough understanding of the pathogenesis of piglet diarrhea and lay the foundation for breeding TGEV‐resistant piglets, but also understanding the molecular mechanisms involved in coronaviral infections.     

用固定细胞阻断酶联免疫吸附试验鉴别诊断猪传染性胃肠炎病毒和猪呼吸道冠状病毒的感染

用固定细胞阻断酶联免疫吸附试验（ＥＬＩＳＡ）对来自丹麦猪的１８０份血清进行了猪传染性胃肠炎病毒（ＴＧＥＶ）和猪呼吸道冠状病毒（ＰＲＣＶ）感染的鉴别诊断，共检出了ＰＲＣＶ抗体阳性血清１０７份（５９．４％），ＴＧＥＶ抗体阳性血清０份。同时也检测了一些来自国内不同ＴＧＥＶ感染类型的猪场血清。该鉴别诊断方法在我国的建立和应用为从ＰＲＣＶ阳性国家进口猪的ＴＧＥ的检疫提供了一条有效途径。     

The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada

Since late 2004, the swine industry in the province of Quebec has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). To explain this phenomenon, 2 hypotheses were formulated: 1) the presence of a 2nd pathogen could be exacerbating the porcine circovirus 2 (PCV-2) infection, or 2) a new and more virulent PCV-2 strain could be infecting swine. In 2005, 13 PMWS cases were submitted to the Quebec provincial diagnostic laboratory and PCV-2 was the only virus that could be found consistently by PCR in all 13 samples. The PCR detection results obtained for other viruses revealed the following: 61.5% were positive for porcine reproductive and respiratory syndrome virus, 30.8% for swine influenza virus, 15.4% for porcine parvovirus, 69.2% for swine torque teno virus (swTTV), 38.5% for swine hepatitis E virus (swHEV) and 84.6% for Mycoplasma hyorhinis; transmissible gastroenteritis virus and porcine respiratory coronavirus (TGEV/PRCV) was not detected. Sequences of the entire genome revealed that these PCV-2 strains belonged to a genotype (named PCV-2b) that has never been reported in Canada. Further sequence analyses on 83 other Canadian PCV-2 positive cases submitted to the provincial diagnostic laboratory during years 2005 and 2006 showed that 79.5% of the viral sequences obtained clustered in the PCV-2b genotype. The appearance of the PCV-2b genotype in Canada may explain the death rate increase related to PMWS, but this relationship has to be confirmed.     

A seroepizootiologic study of five viruses in a swine-evaluation station.

This serologic study was done to gain information on the spread, maintenance, and effect upon performance of five porcine viruses. Blood samples were taken from two groups of 8- to 11-week-old pigs from a large number of Indiana swine herds in a performance-testing station 1 week after entry, 7 weeks after entry (one group only), and at slaughter. The sera were tested by indirect fluorescent antibody tests for antibodies to transmissible gastroenteritis virus (TGEV), swine influenza virus (SIV), hemagglutinating encephalomyelitis virus (HEV), porcine adenovirus (PAV), and pseudorabies virus (PRV). Seroconversions to TGEV, HEV, and PAV occurred in a group of pigs entered in May and slaughtered in August (group 1). In the group that was entered in October and slaughtered in January (group 2), pigs developed antibodies to SIV, HEV, and PAV, but not to TGEV. Only 1 of the 434 pigs tested had antibodies to PRV, and there were no seroconversions to this virus. The only statistically valid effect of infection on performance was found in group 1 pigs, which had seroconverted to TGEV during the first 7 weeks of their stay. These pigs gained 0.077 kg less per day than pigs that did not develop antibodies to TGEV during that period. The pattern of serologic reactions was indicative of a relatively slow spread of these viruses in the groups. We interpret this as supporting the concept that a relatively slow spread of these viruses through large groups of pigs kept under conditions that are less than optimum for virus spread may be an important means of their interepizootic survival.     

贵州省猪传染性胃肠炎及呼吸冠状病毒病血清学调查

用酶联免疫吸附试验对采自贵州省内88个县(市)的2906份血清进行了猪传染性胃肠炎病毒及呼吸冠状病毒抗体检测,结果检出猪传染性胃肠炎病毒抗体阳性血清12份,总体阳性率为0.41%;猪呼吸冠状病毒抗体阳性血清44份,总体阳性率为1.51%;2818份血清两种抗体均呈阴性(阴性率为96.97%),32份血清检测无效或无结论(无效率为1.10%)。     

7种动物冠状病毒特异基因的克隆与序列分析

为了比较冠状病毒基因相关性,获得特异基因克隆制备冠状病毒基因芯片,根据发布的基因序列,每种病毒设计4~17对引物,利用火鸡冠状病毒(TCV)原毒和蔗糖密度梯度离心纯化浓缩的犬冠状病毒(CCV)、猫冠状病毒(FCV)、猫传染性腹膜炎病毒(FIPV)、猪传染性胃肠炎病毒(TGEV)、猪呼吸道冠状病毒(PRCV)、牛冠状病毒(BCV)细胞毒,提取总RNA并反转录和PCR扩增。回收PCR产物连接pGEM-T-easy载体并转化大肠杆菌TGI,经PCR鉴定后测序。将所有基因片段的核苷酸序列和推导的氨基酸序列,分别与GenBank有关病毒相关基因片段的核苷酸序列进行分析比较,确定它们的同源性。通过对不同冠状病毒不同基因片段的克隆和测序,发现同一群冠状病毒核苷酸序列间具有较高的同源性。     

2013-2014年广西省主要猪病毒性传染病流行病学调查

为掌握广西猪主要病毒性传染病流行情况,为猪传染病预防方案提供依据,本研究于2013年1月1日至2014年12月31日从广西省共收集410份样品,运用PCR及RT-PCR方法检测猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV-2)、猪瘟病毒(CSFV)、猪伪狂犬病毒(PRV)、猪流行性腹泻病毒(PEDV)、传染性胃肠炎病毒(TGEV)、轮状病毒(PORV)、猪流感病毒(SIV)的感染情况。检测结果表明,PRRSV、PCV-2、CSFV、PRV、PEDV、TGEV、 PORV和SIV的平均感染率分别为35.12%、18.54%、1.17%、0.98%、10.00%、2.44%、0和1.22%;PRRSV和PCV-2混合感染率为6.83%;PRRSV在秋、冬季节呈现高感染率为36.67%、45.31%和63.64%、48.78%,而PCV-2在春、夏、冬季节呈现高感染率为44.44%、25.00%,11.29%、19.35%和39.39%、14.63%。PRRSV和PCV-2是混合感染的主要病原,它们互相之间或是与CSFV、PEDV、PRV、SIV及副猪嗜血杆菌、链球菌等混合感染,PRRSV和PCV-2将是今后广西地区猪病防控的重点。