Development of Indirect ELISAs for Differential Serodiagnosis of Classical and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

The objective of this study was to develop two indirect enzyme‐linked immunosorbent assays (iELISAs) for detection of serum antibodies against classical vaccine strain of porcine reproductive and respiratory syndrome virus (PRRSV) and highly pathogenic PRRSV (HP‐PRRSV). To detect the common antibodies against classical and HP‐PRRSV, the coating antigen used in the iELISA (designated iELISA‐180) was the antigen of Nsp2‐180, the 180aa at amino terminal of Nsp2. To detect the different antibodies against classical and HP‐PRRSV, the coating antigen in the second iELISA (designated iELISA‐D29) was Nsp2‐D29, the deleted 29aa in Nsp2 of HP‐PRRSV. The antigen concentration and serum dilutions were optimized using a draughtboard titration. The cut‐off values of 0.361 at OD_450nm for the iELISA‐180 and 0.27 at OD_450nm for the iELISA‐D29 were determined by testing a panel of 120 classical PRRSV positive and 198 PRRSV negative pig serum samples, which generated the specificity of 97.1% and 96.7%, the sensitivity of 96.9% and 96.3% for iELISA‐180 and iELISA‐D29, respectively. The agreements between the Western blot and iELISA‐180 and iELISA‐D29 were 98%, 96.7%, respectively. The developed iELISAs can be used to differentiate serologically HP‐PRRSV from the vaccinated or classical PRRSV in clinical serum samples.     

Evolutionary Dynamics of a Highly Pathogenic Type 2 Porcine Reproductive and Respiratory Syndrome Virus: Analyses of Envelope Protein‐Coding Genes

Porcine reproductive and respiratory syndrome virus (PRRSV) has long been an economically devastating swine viral disease. The recent emergence of a highly pathogenic type 2 PRRSV with high mobility and mortality in China, spreading in Vietnam, Laos, and Thailand has placed neighbouring countries at risk. This study applied a codon‐based extension of the Bayesian relaxed clock model and the fixed effects maximum‐likelihood method to investigate and compare the evolutionary dynamics of type 2 PRRSV for all of known structural envelope protein‐coding genes. By comparing the highly pathogenic type 2 PRRSV clade against the typical type 2 PRRSV clade, this study demonstrated that the highly pathogenic clade evolved at high rates in all of the known structural genes but did not display rapid evolutionary dynamics compared with typical type 2 PRRSV. In contrast, the ORF3, ORF5 and ORF6 genes of the highly pathogenic clade evolved in a qualitatively different manner from the genes of the typical clade. At the population level, several codons of the sequence elements that were involved in viral neutralization, as well as codons that were associated with in vitro attenuation/over‐attenuation, were predicted to be selected differentially between the typical clade and the highly pathogenic clade. The results of this study suggest that the multigenic factors of the envelope protein‐coding genes contribute to diversifying the biological properties (virulence, antigenicity, etc.) of the highly pathogenic clade compared with the typical clade of type 2 PRRSV.     

A Bayesian Phylogeographical Analysis of Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

Understanding viral transmission is an important factor for the effective prevention one of the most devastating swine diseases, porcine reproductive and respiratory syndrome. Focusing on molecular epidemiology of type 1 PRRSV, this study analysed a large ORF5 dataset collected worldwide from 1991 to 2012 using a coalescent‐based Bayesian Markov chain Monte Carlo approach. The results suggested that the virus diversified into unique subpopulations in Russia & Belarus and Italy approximately 100 years ago. Previously unreported consecutive diffusions of the virus were identified, which showed that some countries, such as Spain and Germany, acted as distribution sources to some extent. This study also provided statistical evidence for the existence of an ORF5‐based phylogeographical structure of type 1 PRRSV, in which the virus tended to cluster by geographical locations more tightly than expected by chance. In contrast to this tight geographical structure, the evolution of the ORF5 gene, based on mapping of non‐synonymous/synonymous substitutions, was best described by a non‐homogeneous process that could be implicated as a mechanism for viral immune evasion.     

An Outbreak of Porcine Reproductive and Respiratory Syndrome Virus in Switzerland Following Import of Boar Semen

An outbreak of porcine reproductive and respiratory syndrome virus (PRRSV) occurred in November 2012 in Switzerland (CH), traditionally PRRSV‐free. It was detected after a German boar stud informed a semen importer about the detection of PRRSV during routine monitoring. Tracing of semen deliveries revealed 26 Swiss sow herds that had used semen from this stud after its last negative routine monitoring and 62 further contact herds. All herds were put under movement restrictions and examined serologically and virologically. As a first measure, 59 sows from five herds that had previously been inseminated with suspicious semen were slaughtered and tested immediately. Investigations in the stud resulted in 8 positive boars with recent semen deliveries to CH (Seven with antibodies and virus, one with antibodies only). In one boar out of six tested, virus was detected in semen. Of the 59 slaughtered sows, five from three herds were virus‐positive. In one herd, the virus had spread, and all pigs were slaughtered or non‐marketable animals euthanized. In the remaining herds, no further infections were detected. After confirmatory testings in all herds 3 weeks after the first examination gave negative results, restrictions were lifted in January 2013, and Switzerland regained its PRRSV‐free status. The events demonstrate that import of semen from non‐PRRS‐free countries – even from negative studs – poses a risk, because monitoring protocols in boar studs are often insufficient to timely detect an infection, and infections of sows/herds occur even with low numbers of semen doses. The outbreak was eradicated successfully mainly due to the high disease awareness of the importer and because immediate actions were taken before clinical or laboratory diagnosis of a single case in the country was made. To minimize the risk of an introduction of PRRSV in the future, stricter import guidelines for boar semen have been implemented.     

Pathogenicity and Molecular Characterization of Emerging Porcine Reproductive and Respiratory Syndrome Virus in Vietnam in 2007

In 2007, Vietnam experienced swine disease outbreaks causing clinical signs similar to the ‘porcine high fever disease’ that occurred in China during 2006. Analysis of diagnostic samples from the disease outbreaks in Vietnam identified porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV‐2). Additionally, Escherichia coli and Streptococcus equi subspecies zooepidemicus were cultured from lung and spleen, and Streptococcus suis from one spleen sample. Genetic characterization of the Vietnamese PRRSV isolates revealed that this virus belongs to the North American genotype (type 2) with a high nucleotide identity to the recently reported Chinese strains. Amino acid sequence in the nsp2 region revealed 95.7–99.4% identity to Chinese strain HUN4, 68–69% identity to strain VR‐2332 and 58–59% identity to strain MN184. A partial deletion in the nsp2 gene was detected; however, this deletion did not appear to enhance the virus pathogenicity in the inoculated pigs. Animal inoculation studies were conducted to determine the pathogenicity of PRRSV and to identify other possible agents present in the original specimens. Pigs inoculated with PRRSV alone and their contacts showed persistent fever, and two of five pigs developed cough, neurological signs and swollen joints. Necropsy examination showed mild to moderate bronchopneumonia, enlarged lymph nodes, fibrinous pericarditis and polyarthritis. PRRSV was re‐isolated from blood and tissues of the inoculated and contact pigs. Pigs inoculated with lung and spleen tissue homogenates from sick pigs from Vietnam developed high fever, septicaemia, and died acutely within 72 h, while their contact pigs showed no clinical signs throughout the experiment. Streptococcus equi subspecies zooepidemicus was cultured, and PRRSV was re‐isolated only from the inoculated pigs. Results suggest that the cause of the swine deaths in Vietnam is a multifactorial syndrome with PRRSV as a major factor.     

Type 2 Porcine Reproductive and Respiratory Syndrome Virus Infection Mediated Apoptosis in B‐ and T‐Cell Areas in Lymphoid Organs of Experimentally Infected Pigs

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection is characterized by persisting in lungs and lymphoid tissue, resulting in systemic lymphoid depletion. The aim of this study was to correlate the histological changes, viral antigen expression and apoptosis phenomena in tonsil, medial retropharyngeal and mediastinal lymph nodes of 12 pigs inoculated with a type 2 PRRSV isolate (Chilean strain 2402). Apoptosis phenomena were observed mainly in lymphocytes and secondly in macrophages of lymph nodes and tonsils of inoculated animals, showing a peak of both apoptotic cells and viral antigen expression at the end of the study (21 dpi). However, the number of apoptotic cells was higher than the number of PRRSV‐positive cells at the end of the study. This finding together with the location of apoptotic cells and PRRSV‐positive cells in different structures of lymphoid organs supports the hypothesis that PRRSV‐positive macrophages might modulate the apoptosis phenomena in other cells, mainly lymphocytes, by means of an indirect mechanism. Furthermore, apoptotic cells were detected both in B‐ and T‐cell areas of lymphoid organs, suggesting that apoptosis phenomena may play a role in the impairment of the host immune response during PRRS.     

Evaluation of the Pathogenicity and Transmissibility of a Chilean Isolate of Porcine Reproductive and Respiratory Syndrome Virus

The objective of this study was to determine clinical features, shedding and transmission of a Chilean Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) strain upon experimental inoculation of 4‐week‐old pigs. Six groups of five animals each were used. The G1 (donor) group was inoculated with PRRSV, maintained in an isolation unit for 35 days, and sampled daily to determine shedding in mucosal secretions and faeces, viraemia and seroconversion. An uninfected control group (G6) was equally maintained and sampled under strict isolation. Four other groups (G2 to G5) were exposed to PRRSV via direct contact with G1 for 5‐day periods in a staggered manner, throughout the 35‐day period, and were later placed in an independent isolation unit to monitor infection status for 7 days. All the animals in G1 and G6 were killed at 35 days post‐inoculation (dpi) and the contact groups at 12 days post‐contact (dpc). Samples were obtained from diverse organs for histopathological, immunohistochemical (IHC) and virological analysis. No clinical symptoms were evident in any group, except for a transient fever observed in G1. Histopathologically, all the animals of G1 had interstitial pneumonia, although scarce PRRSV‐positive cells were detected in the lung using IHC. PRRSV‐positive cells (IHC) were detected in the lymphoid tissue of all animals in infected groups, but especially in G3 and G4. Viraemia was detected in G1 (3–35 dpi) and in the all contact groups (5–12 dpc). Likewise, ranging from 3 to 19 dpi, PRRSV was detected in at least one animal from the tonsils and lungs in all infected groups, in nasal and ocular secretions, saliva or faeces. These results indicate that the donor group excreted infectious PRRSV and was able to transmit the infection to susceptible pigs. The critical shedding period was 7–19 dpi, during which, most likely, transmission took place.     

Comparative Analysis of Immune Responses in Pigs to High and Low Pathogenic Porcine Reproductive and Respiratory Syndrome Viruses Isolated in China

The CH‐1a and HuN4 strains of porcine reproductive and respiratory syndrome virus (PRRSV) show different pathogenicities in pigs. To understand host immune responses against these viruses, we investigated the dynamic changes in cytokine levels produced in peripheral blood of piglets infected with the highly pathogenic PRRSV HuN4 strain or the CH‐1a strain. Clinical signs, virus loads and serum cytokine levels [interferon(IFN)‐α, Interleukin (IL)‐1, TNF‐α, IL‐6, IL‐12, IFN‐γ, IL‐10 and TGF‐β] were tested. The results showed that while piglets developed effective cellular immune responses against CH‐1a infection, those infected with HuN4 displayed ineffective cellular immunity, organ lesions and persistent elevated levels of immunoregulatory cytokines (IL‐10 and TGF‐β), which delayed the development of PRRSV‐specific immune responses. These results demonstrated that HuN4 infection induced higher cytokine levels than that of CH‐1a infection induced. The changes in inflammatory cytokines intensified the inflammatory reaction and damaged the tissues and organs.     

A recombination between two Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV‐1) vaccine strains has caused severe outbreaks in Danish pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in Danish swine herds. In July 2019, PRRSV‐1 was detected in a PRRSV‐negative boar station and subsequently spread to more than 38 herds that had received semen from the boar station. Full genome sequencing revealed a sequence of 15.098 nucleotides. Phylogenetic analyses showed that the strain was a recombination between the Amervac strain (Unistrain PRRS vaccine; Hipra) and the 96V198 strain (Suvaxyn PRRS; Zoetis AH). The major parent was the 96V198 strain that spanned ORFs 1–2 and part of ORF 3 and the minor parent was the Amervac strain, which constituted the remaining part of the genome. The virus seems to be highly transmissible and has caused severe disease in infected herds despite a high level of genetic identity to the attenuated parent strains. The source of infection was presumable a neighbouring farm situated 5.8 km from the boar station.     

Downregulation of Antigen‐Presenting Cells in Tonsil and Lymph Nodes of Porcine Reproductive and Respiratory Syndrome Virus‐Infected Pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) can persist in different organs of infected pigs, which suggests a failure in the immune response. Antigen‐presenting cells (APCs) play a pivotal role in the induction of effective T‐ and B‐cell responses. In this study, we investigated the changes in the different APC subpopulations and T‐ and B‐cell counts in the tonsil, retropharyngeal and mediastinal lymph nodes of pigs experimentally infected with a European PRRSV field isolate. Our results demonstrated that the expression of S100, SWC3, HLA‐DR molecule and CD3 was diminished in the studied organs throughout the study, observing a significant negative correlation between viral antigen and HLA‐DR expression in both retropharyngeal and mediastinal lymph nodes. In contrast, λ‐light chains showed an increase during the study. Taking all into account, after PRRSV infection, no enhancement in the number of APCs and T cells was observed, suggesting an impairment of the immune function which may allow the persistence of PRRSV into the organism.     

Naturally Co‐Infected Boars with both Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus Type 2

In this study, the humoral response against porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), the presence of the virus in semen and serum and the genetic characteristics of the virus detected in 15 boars from a commercial farm were analysed. The results showed that 53% of the boars presented anti‐PRRSV antibodies and 100% presented anti‐PCV2 antibodies. Porcine reproductive and respiratory syndrome virus was detected in 43% of the boars and 73% were positive to PCV2. The complete ORF5 gene of PRRSV of 14 samples and a fragment of the ORF2 gene of PCV2 of 22 samples were sequenced. Porcine reproductive and respiratory syndrome virus analysis revealed <92% identity in viruses from semen and serum of two boars, whereas in the rest of the boars the identity was >97.5%. As for PCV2, two boars presented an identity <95% in serum and semen and the rest had an identity >96%. The results showed that PRRSV‐ and PCV2‐naturally infected boars can be found, and at least two different strains of viruses from semen and serum can be detected.     

Porcine Reproductive and Respiratory Syndrome Virus Vaccines: Current Status and Strategies to a Universal Vaccine

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, the most significant infectious disease currently affecting swine industry worldwide. In the United States alone, the economic losses caused by PRRS amount to more than 560 million US dollars every year. Due to immune evasion strategies and the antigenic heterogeneity of the virus, current commercial PRRSV vaccines (killed‐virus and modified‐live vaccines) are of unsatisfactory efficacy, especially against heterologous infection. Continuous efforts have been devoted to develop better PRRSV vaccines. Experimental PRRSV vaccines, including live attenuated vaccines, recombinant vectors expressing PRRSV viral proteins, DNA vaccines and plant‐made subunit vaccines, have been developed. However, the genetic and antigenic heterogeneity of the virus limits the value of almost all of the PRRSV vaccines tested. Developing a universal vaccine that can provide broad protection against circulating PRRSV strains has become a major challenge for current vaccine development. This paper reviews current status of PRRSV vaccine development and discusses strategies to develop a universal PRRSV vaccine.     

Epidemiologic and Phylogenetic Characteristics of Porcine Reproductive and Respiratory Syndrome Viruses in Conventional Swine Farms of Jeju Island as a Candidate Region for PRRSV Eradication

Jeju island is the biggest island in Korea. The imports of pigs or their relatives from mainland Korea to this island has been banned since 1998. With this unique geographical and epidemiological context, epidemiology of porcine reproductive and respiratory syndrome virus (PRRSV) was investigated on the island. While all investigated farms showed 100% of seropositive rate for PRRSV, pigs on 37.2% (16/43) of the farms had viremia with type II PRRSV. The seropositive and viremia‐positive rates for PRRSV in 30‐ to 60‐day‐old pigs were significantly higher in the western area (‘swine farm complex’ area) than the eastern area (‘Scattered swine farm’ area) of Jeju island. When 21 ORF5 sequences obtained from viremic sera were phylogenetically analysed, lineage 5 and Kor (newly termed in this study) of type II PRRSV were only found in Jeju island without changes from a previous report (2002–2003). Because other lineages of type II PRRSV (lineage 1 and 3) and type I PRRSV have recently emerged in mainland Korea, the banning of pigs’ movement might be effective to protect the island from the introduction of these new PRRSV genotypes. Under this epidemiological condition (no introduction of new strains except for the modified‐live vaccine (MLV) strain), the positive selection sites were analysed based on ORF5 of the virus. The amino acid 58 of GP5 (located on the hypervariable region) was predicted as a strong positive selection site. Although 51.2% (22/43) of the investigated farms had applied MLV, field strains of type II PRRSV were still circulating with strong positive selection. However, the restricted population of type II PRRSV (lineage 5 and Kor) without introduction of type I PRRSV or the other lineages of type II PRRSV indicate that the island has an effective quarantine system, which would allow PRRSV eradication.     

Emergence of Porcine Reproductive and Respiratory Syndrome in Sweden: Detection,Response and Eradication

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure in sows and respiratory problems in growing pigs. The disease is present in most countries throughout the world but was not diagnosed in Sweden until the summer of 2007 when it was first detected through the national PRRS surveillance program. The immediate mobilization of veterinary authorities, field veterinarians and the pig industry was a prerequisite for preventing the spread of the disease. Within 10 days seven herds were verified as infected and the measures taken included stamping out, cleaning, disinfection and a vacancy period of 3 weeks before the herds were repopulated. To evaluate the effectiveness of these measures, a national sero‐surveillance was carried out during the autumn of 2007. Approximately 90% of the pig production was covered by this screening and all samples tested were negative with regard to antibodies to PRRS virus.     

Differential Expression of Proinflammatory Cytokines in the Lymphoid Organs of Porcine Reproductive and Respiratory Syndrome Virus‐Infected Pigs

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most important diseases in the swine industry. Although several studies have been carried out to elucidate the host immune response evoked against PRRS virus (PRRSV), there are several aspects that still remain unclear. The aim of this study was to determine the expression of IL‐1α, IL‐6 and TNF‐α in the lymphoid organs (mediastinal and retropharyngeal lymph nodes and tonsil) of PRRSV‐infected pigs and to determine their correlation with the expression of PRRSV antigen. Proinflammatory cytokine expression was different depending on the body compartment examined. Thus, whereas IL‐1α and TNF‐α were the main cytokines expressed in the mediastinal lymph node, IL‐6 was the most highly expressed cytokine in the retropharyngeal lymph node, and no expression of proinflammatory cytokines was observed in the tonsil. These findings may be related to the impairment of the host immune response evoked after PRRSV infection. Therefore, lymphoid organs and proinflammatory cytokines represent an important target of study for clarifying the immunopathogenesis of PRRS.     

Fresh Pork and Porcine Reproductive and Respiratory Syndrome Virus: Factors Related to the Risk of Disease Transmission

Porcine reproductive and respiratory syndrome virus (PRRS) is a highly infectious virus. Experimentally, the disease can be induced in naïve pigs by the oral, intranasal and intramuscular routes. Depending on the virulence of the strain of the virus and the age of the pig, peak viremia can occur within 7 days of infection, and live virus can be isolated from blood or lymph nodes for several months post‐infection. Young pigs tend to develop higher titres of viremia than older pigs infected by the same route and dose with the same strain of virus. Porcine reproductive and respiratory syndrome virus survives in pork harvested from infected pigs for extended periods at temperatures of −20 or −70°C. In experimentally infected pigs, survival of PRRS virus in muscle held at 4°C has been demonstrated for at least 7 days, and infectivity of the virus in these samples was confirmed by bioassay. The optimal pH range for the survival of PRRS virus is thought to be 6.0 to 7.5. The elevated pH of non‐meat tissues (generally one pH unit higher) is likely to favour extended survival of PRRS virus in pig carcasses from which all superficial and deep lymph nodes have not been removed. It is likely that exsanguinated carcasses held at 4°C retain sufficient blood or lymph tissue to contain infective doses of PRRS virus. Porcine reproductive and respiratory syndrome virus is rapidly inactivated by heat, providing a predictable method to ensure that pork tissues are free of viable virus and feeding of cooked swill or garbage should not constitute a risk to pigs. While the probability of viable PRRS virus being present in a pig carcass may be low, the risk is not zero. The importation of raw pork into countries where PRRS is not endemic represents a hazard with potentially severe economic consequences.     

Performance of Fattening Pigs in a Farm Infected with Both Porcine Reproductive and Respiratory Syndrome (PRRS) Virus and Porcine Circovirus Type 2 Following Sow and Piglet Vaccination with an Attenuated PRRS Vaccine

The purpose of the study was to investigate whether, on farms with both post‐weaning multisystemic wasting syndrome (PMWS) and porcine reproductive and respiratory syndrome (PRRS), the PRRS vaccination of sows and their fattening pigs protects against these syndromes. In a farrow‐to‐finish pig farm with a history of PRRS and PMWS, 200 gilts and sows were allocated to one of two groups of equal size. The first group (C‐sow group) was used as untreated controls, while the animals of the second group (V‐sow group) were vaccinated with live Porcilis PRRS^® vaccine. At the next weaning, all piglets of half the sows of the C sow group were vaccinated once at 35 days of age with the vaccine (CV group), while the offspring of the other half of the unvaccinated sows were left unvaccinated (CC group). Similarly, the offspring of half the sows of the V sow group were vaccinated (VV group), while those of the other half of the vaccinated sows were left unvaccinated (VC group). No significant differences in morbidity were observed between the groups during the nursery and finishing phases, while morbidity in the growers was significantly reduced in the CV‐ and VV‐groups (P < 0.05). Growers’ mortality was significantly reduced after piglet vaccination when compared with unvaccinated pigs of unvaccinated dams (P < 0.05). Average daily gain and feed conversion ratio were significantly improved in vaccinated piglets compared with those in the unvaccinated groups (P < 0.05).     

Factors affecting Porcine Reproductive and Respiratory Syndrome virus time‐to‐stability in breeding herds in the Midwestern United States

The time needed to wean porcine reproductive and respiratory syndrome (PRRS) virus negative pigs consistently from a breeding herd after an outbreak is referred to as time‐to‐stability (TTS). TTS is an important measure to plan herd closure as well as to manage economic expectations. Weekly PRRS incidence data from 82 sow farms in six production systems located in the Midwestern United States were used for the analysis. The objective of this study was to evaluate the effect of recorded predictors on TTS in participant sow farms. The median TTS was 41.0 weeks (1st quartile 31.0 weeks–3rd quartile 55.0 weeks). In the final multivariable mixed‐effects Cox model, farms that experienced winter (hazard ratio (HR) 2.18, 95% confidence interval (CI) 1.28–3.70) and autumn (HR 1.91, 95% CI 1.16–3.13) PRRS outbreaks achieved stability sooner than farms that experienced PRRS outbreaks during summer. No statistically significant difference (p = 0.76) was observed between the TTS of farms that had a PRRS outbreak during spring and summer (HR 1.09, 95% CI 0.62–1.91). Additionally, farms that had a PRRS outbreak associated with a 1‐7‐4 restriction fragment length polymorphism (RFLP) cut pattern took significantly longer to achieve stability (HR 0.44, 95% CI 0.27–0.72) compared to farms which had a non‐1‐7‐4 PRRS outbreak. Finally, farms that had a previous PRRS outbreak within a year achieved stability sooner (HR 2.18, 95% CI 1.23–3.86) than farms that did not have a previous PRRS outbreak within a year. This study provides information that may result useful for planning herd closure and managing expectations about the time needed to wean PRRS virus negative pigs in breading herds according to the season of the year when the outbreak occurred and the RFLP cut pattern associated with the outbreak virus.