Guangzhi Tong

Highly Pathogenic Porcine Reproductive and Respiratory Syndrome, China

To the Editor: Since April 2006, a highly pathogenic disease caused by unknown agents and characterized by high fever and a high proportion of deaths in pigs of all ages, emerged in some swine farms in Jiangxi Province, People’s Republic of China. The morbidity rate was 50%–100% and mortality rate was 20%–100%. In the next several months, the disease spread rapidly to most provinces of China. In almost all affected swine herds, the following clinical signs were observed: high and continuous fever, anorexia, red discolorations in the bodies, and blue ears; in the late phase of the disease, diarrhea and other clinical signs might be seen due to the secondary infections. Clinical samples (from lungs, kidneys, liver, and lymph nodes) were collected from animals in different provinces and sent for laboratory diagnosis. DNA and RNA were extracted from the tissue homogenate and PCR or reverse transcription–PCR (RT-PCR) was conducted to detect porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus, porcine circovirus, and pseudorabies virus, respectively (1). In clinical samples, only PRRSV was found to be the dominant virus (48 of 50 samples were PRRSV positive). PRRSVs were then isolated successfully on MARC-145 cells with an obvious cytopathologic effect, characterized by cell congregation, contraction, and brushing off at passage 2; immunofluorescence assay using PRRSV NP-, M- and GP5-specific monoclonal antibodies confirmed that the isolated viruses were PRRSV (2,3). Full-length genomic sequencing of 1 of the isolates (HuN4 strain) showed extensive amino acid (aa) mutations in GP5 protein and 2 deletions in Nsp2, 1 aa deletion at 482, and 29 aa deletions at 533–561, compared with the previous Chinese isolates CH-1a and BJ-4. The newly isolated PRRSV was used to examine the pathogenicity in 60-day-old PRRSV-free piglets, under closed and biosafety (P2) conditions. Each of the piglets (N = 5) received intranasally 105.0 50% tissue culture infecting dose of the isolated virus propagated in MARC-145 cells (4,5). The animals were kept in separate rooms throughout the experiment. Clinical observations of respiratory signs, behavior, rectal temperature, and coughing were recorded daily. Blood samples were collected every 2 days and tested for PRRSV-specific antibodies by ELISA (6,7). Tissue samples (from heart, lungs, kidneys, spleen, and lymph nodes) from all animals that died during the experiment were collected and detected by histopathologic examination (8) and virus isolation. Results showed that the clinical manifestations of all pigs were similar to those that appeared in the field investigation (including high and continuous fever, anorexia, red discolorations in the bodies, and blue ears). The specific antibodies to PRRSV were detected at 8 days postinfection, and the high antibody level lasted until the animal’s death, and all infected pigs died at either 7, 8, 12, 16, or 21 days postinoculation, respectively. Furthermore, viruses reisolated from the dead pigs showed an identical homology with the inoculated PRRSV in genes coding for GP5 and partial Nsp2 (2,535–3,307 nt). The results showed that the emerging PRRSV, characterized by deletions in Nsp2, is highly pathogenic to pigs. To investigate whether the emerging PRRSV was the causative agent of the pandemic diseases on swine farms, an extensive virus survey was conducted. More than 48 samples collected from different swine farms in12 provinces were found to be PRRSV positive by RT-PCR, based on open reading frame (ORF) 5 and Nsp2 (Figure). Sequence analysis of ORF5 and partial Nsp2 showed that these PRRSVs are highly homologous to each other (98.5%–100% for GP5; 98.2%–100% for Nsp2) and share the same deletions at the same positions of Nsp2 gene with HuN4 strain. Sequence comparison of ORF5 indicated that the HuN4 strain shares 93%, 86%, and 88% nucleotide identities with CH-1a (Chinese isolate), BJ-4 (Chinese isolate), and VR2332 (American isolate), respectively. All the newly isolated PRRSVs belong to the North American type. Figure Geographic distribution of porcine reproductive and respiratory syndrome viruses (PRRSVs) examined in the study. Shaded areas indicate the provinces where the PRRSVs characterized by deletions in Nsp2 were detected. Although the cause of the emerging pandemic disease of pigs with a high proportion of deaths in 2006 is unknown, we found high correlation between PRRSV isolation rate and the diseased pigs. The regression test in its natural animal showed that the newly isolated PRRSV was much more virulent than earlier PRRSV isolates. Also, sequence analysis demonstrated a substantial diversity from the PRRSVs isolated during 1996–2005. Further study is needed to answer the question: What role did the newly isolated PRRSV play in the 2006 outbreaks on many of the swine farms in China?

Highly Virulent Porcine Reproductive and Respiratory Syndrome Virus Emerged in China

A highly pathogenic pig disease emerged in China in 2006, which was characterized by prolonged high fever, red discoloration of the body, and blue ears associated with high mortality. Porcine reproductive and respiratory syndrome virus (PRRSV) was isolated as the single most prominent virus in the samples collected from affected pigs. The full-length genomic sequence of the virus revealed two distinct deletions in the non-structural protein 2 (NSP2) in comparison to all previously reported North American genotype PRRSV. Through extensive surveys in 14 different provinces, 56 additional PRRSV isolates were obtained from affected farms. All of the isolates were found to contain identical deletions in NSP2. To confirm the etiology, eight 60-day-old PRRSV-free pigs were divided into two groups and the test group was intranasally infected at a titer of 2 x 10(5.0) tissue culture infectious dose 50 per pig. The inoculated pigs all died at 7, 8, 12, 16, or 21 days post-inoculation with their clinical and pathological findings similar to those in the field. The viruses recovered from dead pigs were identical to the inoculated virus in NSP2 and GP5 genes. Our study shows that the recently emerged PRRSV in China is characterized by two discontiguous deletions in NSP2 and is the cause for the current epizootics in China.

Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein

Abstract S1D (residues 636–789) is a neutralizing epitope region on the spike protein (S) of porcine epidemic diarrhea virus (PEDV). To accurately identify epitopes on S1D, the S1-phage library containing the gene encoding the S1D region of PEDV S protein was micropanned by six specific monoclonal antibodies (McAbs) against the S1D region. These micropanned epitope regions (MER) were focused on 696–779 amino acids of the S protein. To further map epitopes of the MER, seven overlapping mini-fragments covering MER nucleotides were separately synthesized and expressed in Escherichia coli BL21 with a GST tag. These mini-GST fusion proteins were scanned by ELISA and Western blotting with the six McAbs, and the result showed that S1D5 (residues 744–759) and S1D6 (residues 756–771) are two linear epitopes of the PEDV S protein. The antisera of the epitopes S1D5 and S1D6 could react with the native S protein of PEDV. Furthermore, Pepscan of the two linear epitopes demonstrated that SS2 (748YSNIGVCK755) and SS6 (764LQDGQVKI771) are two core epitopes on S1D5 and S1D6, respectively, located on the S protein of PEDV.

An infectious disease of ducks caused by a newly emerged Tembusu virus strain in mainland China.

During investigations into an outbreak of egg production decline, retarded growth, and even death among ducks in Southeast China, a novel Tembusu virus strain named Tembusu virus Fengxian 2010 (FX2010) was isolated. This virus replicated in embryonated chicken eggs and caused embryo death. In cross-neutralization tests, antiserum to the partial E protein of Tembusu virus Mm1775 strain neutralized FX2010, whereas antiserum to Japanese encephalitis virus did not. FX2010 is an enveloped RNA virus of approximately 45-50 nm in diameter. Sequence analysis of its E and NS5 genes showed that both genes share up to 99.6% nucleotide sequence identity with Baiyangdian virus, and up to 88% nucleotide sequence identity with their counterparts in Tembusu virus. FX2010 was transmitted without mosquito, and caused systemic infection and lesions in experimentally infected ducks. These results indicate that FX2010 and BYD virus are newly emerged Tembusu virus strains that cause an infectious disease in ducks.

Genetic diversity of avian infectious bronchitis coronavirus strains isolated in China between 1995 and 2004

Summary.Twenty-six avian infectious bronchitis (IB) viruses (IBV) were isolated from outbreaks in chickens in China between 1995 and 2004. They were characterized by comparison with twenty-six Chinese reference strains and five other IBV strains. Chinese IBVs, which were mainly nephropathogenic, were placed into seven genotypes. Fourteen Chinese IBV isolates were placed in genotype I, having small evolutionary distances from each other. Genotype II included 6 strains that were isolated in the 1990s in China. Genotype III consisted of eight Chinese isolates that showed close relationship with Korean IBV isolates. Another eight IBV isolates clustered in genotype IV and showed larger evolutionary distances. The Massachusetts serotype was present in China in 1990s and was in a separate genotype. Two isolates, HN99 and CK/CH/LHN/00I, which might be a reisolation of vaccine strains, clustered into genotype VI. Four Chinese IBV isolates formed another genotype and showed larger evolutionary distances from other Chinese IBV genotypes (genotype VII). IBVs in same genotypes showed more than 90% amino acid sequence similarities, whereas most of the viruses in different genotypes showed less than 90%. The results showed that IBVs in China came from genetic changes both in IBV populations that existed before the advent of vaccination and in the viruses that were introduced through live vaccines. IBVs showing various genetic differences are cocirculating in China.

An attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) protects piglets against HP-PRRS

Porcine infections with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) cause significant morbidity and mortality and currently there are no effective vaccines for disease prevention. An attenuated strain, HuN4-F112, was obtained by passaging the HP-PRRSV HuN4 on Marc-145 cells (112th-passage). PRRSV-free pigs were inoculated intramuscularly with HuN4-F112 (10(2.0), 10(3.0), 10(4.0), 10(5.0) and 10(6.0) TCID(50) for groups 1-5, respectively). The groups 3-5 could resist the lethal challenge and did not show any obvious changes in body temperature nor clinical signs throughout the experiment, the pathological lesions were milder and the gained weight at a greater rate (P<0.05), compared to group 1 and control. Sequence analysis of the HuN4 passages showed a conserved epitope in GP5 protein was mutated ((196)QWGRL/P(200)-->(196)RWGRL/P(200)), as a result the monoclonal antibody could not recognize the HuN4-F112 any more. These results suggested that the HuN4-F112 could protect piglets from lethal challenge and might be a candidate vaccine against the HP-PRRSV.

Pseudorabies Virus Variant in Bartha-K61-Vaccinated Pigs, China, 2012

The widely used pseudorabies virus (PRV) Bartha-K61 vaccine has played a key role in the eradication of PRV. Since late 2011, however, a disease characterized by neurologic symptoms and a high number of deaths among newborn piglets has occurred among Bartha-K61–vaccinated pigs on many farms in China. Clinical samples from pigs on 15 farms in 6 provinces were examined. The PRV gE gene was detectable by PCR in all samples, and sequence analysis of the gE gene showed that all isolates belonged to a relatively independent cluster and contained 2 amino acid insertions. A PRV (named HeN1) was isolated and caused transitional fever in pigs. In protection assays, Bartha-K61 vaccine provided 100% protection against lethal challenge with SC (a classical PRV) but only 50% protection against 4 challenges with strain HeN1. The findings suggest that Bartha-K61 vaccine does not provide effective protection against PRV HeN1 infection.

Highly pathogenic porcine reproductive and respiratory syndrome virus, Asia.

To the Editor: Recently, the novel and highly virulent variant of porcine reproductive and respiratory syndrome virus (PRRSV), which first emerged in the People’s Republic of China and Vietnam in 2006 (1), has rapidly spread in pigs in Southeast Asia. The affected countries include Bhutan, Cambodia, Laos, Malaysia, Myanmar, the Philippines, Thailand, and Singapore. In eastern and northern Asia, South Korea and Russia were also reported to be affected (2) (Figure). The epidemic affected not only large commercial farms but also the backyard industry, which created a serious problem for the global swine industry and for food safety. In February 2011, the Veterinary and Animal Breeding Agency in Ulaanbaatar, Mongolia, confirmed an outbreak of porcine reproductive and respiratory syndrome (PRRS) (3). Nearby neighbors, such as Japan, North Korea, Indonesia, and other Asia-Pacific countries, are also at risk. Figure Areas in Asia where outbreaks of highly pathogenic porcine reproductive and respiratory virus syndrome occurred. The countries or regions affected (North Asia, East Asia, Asia, and South Asia) are indicated. PRRS was first reported in the United States in 1987. The disease causes reproductive failure during late-term gestation in sows and respiratory disease in pigs of all ages. In 2006, a new, highly pathogenic PRRS emerged, characterized by high fever (41°C–42°C), skin discoloration/reddening, high incidence of illness (50%–100%), and high proportion of deaths (20%–100%) in pigs of all ages. This new PRRS has spread throughout the swine industry in China, resulting in the culling of an estimated 20 million pigs annually in 2006–2007 in China (4). PRRSV is a member of the family Arteriviridae in the order Nidovirales, which also includes severe acute respiratory syndrome coronavirus. PRRSV is a single-stranded positive sense RNA virus that shows high rates of genetic diversity. In the genome of the novel highly pathogenic PRRSV mutant, 4 deletions (2 deletions in nonstructural protein 2, one deletion in the 5′ untranslated region, and one deletion in the 3′ untranslated region), and some other point mutations, have occurred, which were markedly different from those found in any other previous virus isolate. After a surveillance study of the epidemic and an analysis of >300 novel highly pathogenic PRRSVs were conducted, the highly pathogenic PRRSV from China was considered to have gradually evolved from CH-1a, a local PRRSV isolate. The evolutionary path could be traced through intermediate PRRSV strains (5). Moreover, we found that highly pathogenic PRRSV has a further enlarged deletion in nonstructural protein 2. Highly pathogenic PRRSV first emerged in China and Vietnam almost simultaneously in 2006, and the epidemic focus was in the area between southern China and northern Vietnam (6,7). Although no evidence has shown that the highly pathogenic PRRSV isolate from China or Vietnam has spread in other areas, highly pathogenic PRRS has spread throughout the Malaysian Peninsula to southern Russia. In addition, all highly pathogenic PRRSV isolates share high sequence identity and have the same deletions as the highly pathogenic PRRSV isolated from China or Vietnam. PRRSV can spread through a variety of routes, including direct contact between pigs, droplet contact through nasal secretions, direct contact with saliva and feces, and indirect contact. PRRS has spread rapidly around the world through pig sales, semen, and airborne transmission, including from airline passengers who carry the virus on their clothing, shoes, or equipment while traveling (8). In the global market, any virus emerging in the highly pathogenic form is a threat. The risk of highly pathogenic PRRS spreading to other countries is increasing.

Genetic Evolution of Swine Influenza A (H3N2) Viruses in China from 1970 to 2006

ABSTRACT 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 wholly human-like H3N2 viruses, double-reassortant H3N2 viruses, and triple-reassortant H3N2 viruses in pigs in China by analyzing the eight genes of swine influenza A (H3N2) viruses found in China from 1970 to 2006. In 1970, the first wholly human-like H3N2 (Hong Kong/68-like) viruses were isolated from pigs in Taiwan, and then in the next years Victoria/75-like, Sydney/97-like, New York/99-like, and Moscow/99-like swine H3N2 viruses were regularly isolated in China. In the 1980s, two triple-reassortant viruses were isolated from pigs. Recently, the double-reassortant viruses containing genes from the human (HA and NA) and avian (PB2, PB1, PA, NP, M, and NS) lineages and the triple-reassortant viruses containing genes from the human (HA and NA), classical swine (NP), and avian (PB2, PB1, PA, M, and NS) lineages emerged in pigs in China. The coexistence of wholly human-like and reassortant viruses provides further evidence that pigs serve as intermediate hosts, or mixing vessels, and emphasizes the importance of reinforcing swine influenza virus surveillance in China.

Origin of highly pathogenic porcine reproductive and respiratory syndrome virus, China.

To the Editor: A highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), which affected >2 million pigs, emerged in early 2006 in the People’s Republic of China. The disease was characterized by high fever (41°C), high illness rates (50%–100%), and high death rates (20%–100%) for pigs of all ages (1). A number of HP-PRRSVs have been isolated from 2006 through 2009 from infected pigs in different provinces of China and confirmed to be the causative agent of the new outbreaks (1,2). These HP-PRRSVs have a deletion of 30 amino acids in the nonstructural 2 (NSP-2). However, the evolutionary origin and path of the HP-PRRSV remain unknown. We analyzed the full-length sequences of 67 PRRSVs: 35 HP-PRRSVs (HuN4 and LNSY-08-1 isolated in our laboratory and 33 viruses isolated in other laboratories), 28 classic PRRSVs (18 viruses isolated from China and 10 viruses representing other Asian countries and North America), and 4 commercially available attenuated live PRRSV vaccine viruses. Except for the 2 viruses we isolated (HuN4 and LNSY-08-1), the full-length sequences of the other 65 viruses were obtained from GenBank. Nucleotide and deduced amino acid sequences of these PRRSVs were aligned and compared by using previous methods (3,4). Whole genome–based phylogenetic analysis showed that these 67 PRRSVs could be divided into 4 subgroups (Appendix Figure). Ten classic PRRSVs from China, together with the North American prototype virus VR-2332 and the vaccine virus RespPRRS/Repro modified live vaccine, were classified into subgroup 1. The first Chinese isolate, CH-1a, and its 3 derivatives (CH2002, CH2003, and CH2004) were classified into subgroup 2. All 35 HP-PRRSVs were classified into subgroup 4, and they shared high homology (>99%) in their genomic sequences. The other 4 Chinese PRRSVs, including HB-1(sh)/2002, HB-2(sh)/2002, Em2007, and SHB, belonged to subgroup 3, an intermediate subgroup between subgroups 2 and 4. Phylogenetically, HP-PRRSVs had a close relationship with subgroups 2 and 3. Four conserved deletions were shown among all HP-PRRSVs, including an adenosine deletion at position 122 in the 5′-untranslated region, a guanosine deletion at position 15,278 in the 3′-untranslated region, and 2 discontinuous deletions in the NSP-2, including a single amino acid deletion at position 482 (L482) and a second deletion of 29 amino acids between positions 533 and 561 (S533–A561). The presence of these 4 deletions among subgroup 4 viruses is a unique phenomenon, which may be used as a distinctive molecular marker for HP-PRRSVs. The occurrence of these 4 deletions might be explained as a stepwise accumulation from subgroup 2 to subgroup 4. None of the 4 deletions were found in subgroup 2. Among viruses in subgroup 3, one, 2, or 3 of the 4 deletions occurred. For example, a single deletion was present at 122 nt in Em2007, double deletions at 122 nt and 15,278 nt in HB-1(sh)/2002 and SHB, and triple deletions at 122 nt, 15,278 nt, and 482 aa in GD3-2005 (this sequence was not submitted to GenBank until now). In 2008, Ma et al. compared GD3-2005 with several PRRSVs and reported the homology within them, pointing out that the 2 deletions in NSP-2 were identical to the HP-PRRSV (5). After careful analysis, we found the GD3-2005 more interesting than what was reported in Ma et al.; it belongs to an intermediate group, and shares the characters of gradual evolution. Eventually, all 4 deletions occurred in subgroup 4. This obvious pattern suggests that these 4 conserved deletions might have evolved step by step. The primary neutralizing epitope (PNE), which is located on glycoprotein 5 and composed of the residues S37H(F/L)QLIYN with F/L39 as the binding site for the neutralizing antibody (6,7), also displayed similar changes at the 39 position among the 4 subgroups. The PNE residues in subgroups 1 (SHL39QLIYN) and 2 (SHF39QLIYN) were considerably conservative. Subgroup 3 contained either F39 or I39 (F39 in Em2007 and HB-2(sh)/2002, and I39 in both HB-1(sh)/2002 and SHB); subgroup 4 contained I39 only. The existence of either F39 or I39 in subgroup 3 PNE indicates its intermediate position between subgroups 2 and 4 in the evolution of HP-PRRSVs. Pairwise comparison of subgroups 2, 3, and 4 did not find recombination or large fragment replacement, which suggests that all HP-PRRSVs originated from the same ancestor by gradual evolution. Notably, the recently isolated intermediate PRRSVs mentioned above (SHB, Em2007, and GD3-2005) were isolated in the region of South China where the outbreak of HP-PRRS initially occurred. Furthermore, the epidemiologic data show that the outbreak of HP-PRRSV emerged from 1 particular place and then spread widely. This evidence indicates that all HP-PRRSVs isolated in China likely originated from the same source. In summary, our findings suggest that the newly emerged HP-PRRSVs originated from the Chinese CH-1a-like PRRSV. Further study is needed to determine what contributes to the increased pathogenicity of HP-PRRSV. Although the 4 deletions are conserved in all HP-PRRVs, the increased pathogenicity of HP-PRRSV may not merely be caused by the deletions; pathogenicity is affected by multigenetic factors.