Comparison of individual, group and environmental sampling strategies to conduct influenza surveillance in pigs.

BACKGROUND: Influenza A virus (IAV) is an important pathogen in pigs that affects productivity and has important public health implications because of its zoonotic nature. Surveillance is central to the control of influenza, however, detection of IAV infections can be challenging in endemically infected herds with low prevalence of infection. METHODS: In groups of suckling (18-21 days of age) and growing (35-45 days of age) pigs, we compared various sampling approaches to detect, isolate and sequence IAV using individual (nasal swabs, nasal wipes and oropharyngeal swabs), group (oral fluids, surface wipes and sow udder skin wipes) and environmental (airborne particles deposited on surfaces and air samples) sampling approaches. All samples were tested by IAV rRT-PCR and a subset was used for virus isolation and direct sequencing. RESULTS: In general, environmental and group samples resulted in higher odd ratios (range = 3.87-16.5, p-value < 0.05) of detecting a positive sample by rRT-PCR compared to individual pooled samples, except for oropharyngeal swabs (OR = 8.07, p-value < 0.05). In contrast, individual samples were most likely to yield a viral isolate by cell culture. Oropharyngeal swabs in suckling pigs (78.4%), and nasal swabs (47.6%) or nasal wipes (45%) in growing pigs, and udder wipes in lactating sows (75%) were the preferred samples to obtain an isolate. CONCLUSIONS: Our findings indicate that group and environmental sampling strategies should be considered in influenza surveillance programs in particular if the goal is just to detect infection. This study provides new information on sampling approaches to conduct effective influenza surveillance in pigs and identifies udder wipes from lactating sows as a novel sample type that offers a convenient, cheap and sensitive manner to monitor IAV in litters prior to weaning.

Detection and genetic characterization of porcine astroviruses in piglets with and without diarrhea in Thailand.

Porcine astrovirus (PAstV) is widely distributed and highly prevalent among pigs, nevertheless its clinical significance remains unclear as it can be detected in both diarrheic and in healthy pigs. Information about the prevalence, clinical significance and molecular characterization of PAstV in Thailand is not available. This study investigated the prevalence of PAstV in 488 fecal samples collected from piglets with and without diarrhea in 28 pig farms in northern and central parts of Thailand using RT-PCR. The overall prevalence of PAstV infection was 6.5% (32/488), of which 21/251 (8.4%) were in diarrheic and 11/237 (4.6%) were in healthy pigs. Of 32 positive samples, 46.9% were positive for PAstV alone whereas 53.1% were co-infected with porcine group A rotavirus (PRVA). A phylogenetic analysis of the partial RNA-dependent RNA polymerase/capsid genes revealed two lineages of PAstV strains detected in this study. PAstV4 was the most dominant genotype (92%), followed by PAstV2 (8%). This study revealed for the first time that PAstV4 and PAstV2 were circulating in Thailand with PAstV4 as the most dominant genotype in pig herds in northern and central parts of Thailand.

Influenza Transmission in the Mother-Infant Dyad Leads to Severe Disease, Mammary Gland Infection, and Pathogenesis by Regulating Host Responses.

Seasonal influenza viruses are typically restricted to the human upper respiratory tract whereas influenza viruses with greater pathogenic potential often also target extra-pulmonary organs. Infants, pregnant women, and breastfeeding mothers are highly susceptible to severe respiratory disease following influenza virus infection but the mechanisms of disease severity in the mother-infant dyad are poorly understood. Here we investigated 2009 H1N1 influenza virus infection and transmission in breastfeeding mothers and infants utilizing our developed infant-mother ferret influenza model. Infants acquired severe disease and mortality following infection. Transmission of the virus from infants to mother ferrets led to infection in the lungs and mother mortality. Live virus was also found in mammary gland tissue and expressed milk of the mothers which eventually led to milk cessation. Histopathology showed destruction of acini glandular architecture with the absence of milk. The virus was localized in mammary epithelial cells of positive glands. To understand the molecular mechanisms of mammary gland infection, we performed global transcript analysis which showed downregulation of milk production genes such as Prolactin and increased breast involution pathways indicated by a STAT5 to STAT3 signaling shift. Genes associated with cancer development were also significantly increased including JUN, FOS and M2 macrophage markers. Immune responses within the mammary gland were characterized by decreased lymphocyte-associated genes CD3e, IL2Ra, CD4 with IL1ß upregulation. Direct inoculation of H1N1 into the mammary gland led to infant respiratory infection and infant mortality suggesting the influenza virus was able to replicate in mammary tissue and transmission is possible through breastfeeding. In vitro infection studies with human breast cells showed susceptibility to H1N1 virus infection. Together, we have shown that the host-pathogen interactions of influenza virus infection in the mother-infant dyad initiate immunological and oncogenic signaling cascades within the mammary gland. These findings suggest the mammary gland may have a greater role in infection and immunity than previously thought.

Increased frequency of porcine epidemic diarrhea virus shedding and lesions in suckling pigs compared to nursery pigs and protective immunity in nursery pigs after homologous re-challenge.

Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs and spreads rapidly after entering naïve pig populations. The objectives were to (1) compare the disease course following inoculation with PEDV isolate US/Colorado/2013 in naïve 10 day and 8 week-old pigs, and (2) contrast the naïve response to homologous challenge in 8 week-old pigs. Pigs were randomly assigned into group 1 (n = 40, no PEDV exposure), group 2 (n = 43, PEDV inoculation at 10 days of age) and group 3 (n = 48, PEDV inoculation at 8 weeks of age). Thirty-three group 2 pigs received a homologous challenge at 8 weeks of age. Following primary or secondary inoculation, 3-10 pigs were euthanized at days post-inoculation (dpi) 1, 2, 3, 7 or 14. Clinical signs were more pronounced in 10 day-old pigs compared to 8 week-old pigs at dpi 2 and 3, a higher number of 10 day-old pigs shed PEDV RNA in feces compared to 8 week-old pigs. Typical severe atrophic enteritis of PEDV infection was observed at dpi 3 in both age groups, and at dpi 4 and 14 fecal shedding patterns were also similar. While both age groups had seroconverted to PEDV by dpi 14, IgG levels were higher in 8 week-old pigs. PEDV IgA antibodies were detected in feces of approximately 50% of the pigs at dpi 44. In homologous challenged pigs, no clinical signs or lesions were found, and PEDV fecal shedding was restricted to less than 10% of the pigs indicating the existence of homologous protection 44 days after initial PEDV exposure.

Jaagsiekte Sheep Retrovirus Can Reach Peyer's Patches and Mesenteric Lymph Nodes of Lambs Nursed by Infected Mothers.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung cancer of sheep caused by jaagsiekte sheep retrovirus (JSRV). It is generally accepted that transmission by the respiratory route occurs under natural conditions. However recent studies strongly indicate that JSRV can also be transmitted to lambs perinatally via colostrum and milk (C/M). The aim of this work was to confirm that C/M can transmit JSRV infection to lambs under natural conditions and investigate the initial events associated with this transmission route. We have analyzed the presence of JSRV in C/M samples from 22 naturally infected, asymptomatic ewes throughout a lactation period, and in various tissues collected from a group of 36 of their lambs that were fed naturally. The lambs were euthanized at 12, 24, 48, and 72 hours and at 5 and 10 days after birth. We detected JSRV-provirus by PCR in the somatic C/M cells from 10/22 ewes (45.45%). The virus was also detected in 9/36 lambs (25%). JSRV-infected cells, with lymphoreticular-like morphology, were observed by immunohistochemistry (IHC) and in situ hybridization (ISH) in Peyer's patches (PP) from the small intestine of the youngest lambs and in mesenteric lymph nodes (MLN) from lambs older than 72 hours. The virus was also detected by PCR in white blood cells (WBC) in 2/36 lambs (5.5%). These results confirm colostral transmission of JSRV to lambs under natural conditions. Infected lymphoreticular cells contained in C/M appear to be involved. These cells can cross the intestinal barrier of newborn lambs, reach the MLN and enter into circulation.

Infection of farmed pigs with porcine kobuviruses in Italy.

Two-hundred eight swine fecal samples from six Italian farms were tested using a kobuvirus-specific RT-PCR with primers that amplify a region within the 3D gene. All farms were kobuvirus positive, with prevalence rates ranging between 24 % and 84 %. Overall, 57.5 % of asymptomatic pigs and 49.7 % of animals with diarrhea were positive for kobuvirus. Sequence analysis showed a different predominant strain circulating on each farm and indicated that the strains detected were related to both European and Asiatic strains. A possible pathogenic role of kobuvirus should be investigated further, since infections with this virus occur frequently in pigs of different ages.

Implication of Hsc70, PDI and integrin αvß3 involvement during entry of the murine rotavirus ECwt into small-intestinal villi of suckling mice.

In the present study, a homologous rotavirus, ECwt, infecting small intestinal villi isolated from ICR and BALB/c mice were used as a model for identifying cell-surface molecules involved in rotavirus entry. Small-intestinal villi were treated with anti-Hsc70, anti-PDI, anti-integrin ß3 or anti-ERp57 antibodies or their corresponding F(ab')2 fragments before inoculation with rotavirus ECwt, RRV or Wa. Pretreatment of villi decreased virus infectivity by about 50-100 % depending of the rotavirus strain, antibody structure and detection assay used. Similar results were obtained by treating viral inocula with purified proteins Hsc70, PDI or integrin ß3 before inoculation of untreated villi. Rotavirus infection of villi proved to be sensitive to membrane-impermeant thiol/disulfide inhibitors such as DTNB and bacitracin, suggesting the involvement of a redox reaction in infection. The present results suggest that PDI, Hsc70 and integrin ß3 are used by both homologous and heterologous rotaviruses during infection of isolated mouse villi.

Prevalence of antibodies against transmissible gastroenteritis virus and porcine respiratory coronavirus among pigs in six regions in Japan.

A total of 2,703 pig sera from 171 farms in six regions in Japan were screened for virus-neutralizing (VN) antibody against transmissible gastroenteritis virus (TGEV). Although none of the farms had clinical signs of transmissible gastroenteritis (TGE) or vaccination against TGEV, VN antibody was detected in 14.4% of sera at 30 farms (17.5%) across all six regions. On testing of 263 VN antibody-positive sera from 27 farms with a commercial blocking ELISA to distinguish TGEV and porcine respiratory coronavirus (PRCV) antibodies, 78.3% were positive for PRCV antibody only, while 12.5% were positive for TGEV antibody only or both TGEV and PRCV antibodies. Seven of the eight TGEV antibody-positive farms were also positive for PRCV antibody. Five months after the antibody examination, a TGE outbreak occurred at one of these seven farms. These results suggest that most of the detected VN antibodies were to PRCV, and that TGEV infection might be present at some PRCV-positive farms in Japan.

Efficacy of arbidol on lethal hantaan virus infections in suckling mice and in vitro.

AIM: Arbidol is an immunomodulator that was first developed in Russia. In this study, we report the antiviral activity of arbidol against Hantaan virus (HTNV) in vitro and in vivo. METHODS: The antiviral activity of arbidol in vitro was determined by plaque-forming assay, ranging from 0.5 to 8 microg/mL. To investigate whether arbidol has an antiviral effect in vivo, suckling BALB/c mice infected with HTNV were treated with arbidol at 24 h before infection with a 5, 10 or 20 mg.kg(-1).d(-1), once per day, for 10 days. On day 12 and 28 post infection (pi), histopathological changes and viral antigen were detected. On days 4, 8, 12, and 16 pi, the viral load of target organs and serum TNF-alpha levels of arbidol-treated animals were determined. RESULTS: Arbidol was found to have potent inhibitory activity against HTNV when added in vitro before or after viral infection, with a 50% inhibitory concentration (IC(50)) of 0.9 and 1.2 microg/mL, respectively. The 50% lethal dose (LD(50)) of arbidol for suckling mice was 78.42 mg.kg(-1).d(-1). Oral administration of arbidol increased both survival rate and mean time to death (MTD). Treatment with arbidol reduced histopathological changes, decreased viral load and viral antigen levels, and modulated the level of serum TNF-alpha. CONCLUSION: Arbidol has the ability to elicit protective antiviral activity against HTNV in vivo and in vitro.Acta Pharmacologica Sinica (2009) 30: 1015-1024; doi: 10.1038/aps.2009.53; published online 8 June 2009.

Epitope mapping and cellular localization of swine acute diarrhea syndrome coronavirus nucleocapsid protein using a novel monoclonal antibody.

A swine acute diarrhea syndrome coronavirus (SADS-CoV) that causes severe diarrhea in suckling piglets was identified in Southern China in 2017. To develop an antigen that is specific, sensitive, and easy to prepare for serological diagnosis, antigenic sites in the SADS-CoV nucleocapsid (N) protein were screened. We generated and characterized an N-reactive monoclonal antibody (mAb) 3E9 from mice immunized with recombinant N protein. Through fine epitope mapping of mAb 3E9 using a panel of eukaryotic-expressed polypeptides with GFP-tags, we identified the motif 343DAPVFTPAP351 as the minimal unit of the linear B-cell epitope recognized by mAb 3E9. Protein sequence alignment indicated that 343DAPVFTPAP351 was highly conserved in different SADS-CoV strains and SADS-related coronaviruses from bat, with one substitution in this motif in HKU2-related bat coronavirus. Using mAb 3E9, we observed that N protein was expressed in the cytoplasm and was in the nucleolus during SADS-CoV replication. N protein was immunoprecipitated from SADS-CoV-infected Vero E6 cells. Taken together, our results indicated that 3E9 mAb could be a useful tool to investigate the structure and function of N protein during viral replication.

Excretion of hepatitis E virus by pigs of different ages and its presence in slurry stores in the United Kingdom.

Five faecal samples were collected from four different stages of production at each of 10 pig farms in the Yorkshire Humberside area of the UK, and samples of slurry were collected from nine of the farms. All the samples were tested for hepatitis E virus (HEV) RNA by a nested reverse transcriptase PCR. At least one sample from the pigs on each of the farms tested positive for hev; its prevalence in the 10 herds varied from 5 per cent to 35 per cent and its mean prevalence was 21.5 per cent. The mean prevalence in pigs aged three to five weeks was 26.0 per cent, in pigs aged 10 to 12 weeks 44.0 per cent, in pigs aged 22 to 24 weeks 8.9 per cent, and in adult dry sows 6.0 per cent. Two of the nine slurry lagoons tested positive for HEV RNA. Phylogenetic analysis of the sequence data indicated that the strains of the virus were of genotype 3 and closely related to strains detected in other pigs and in human beings in the UK.

Detection of atypical porcine pestivirus in Brazil in the central nervous system of suckling piglets with congenital tremor.

Atypical porcine pestivirus (APPV) has been detected in piglets with congenital tremor (CT) from three different continents including North America, Europe and Asia. Thirteen piglets from four farms in two different states in Brazil with CT were sampled. Viral RNA was detected by quantitative real-time PCR in the cerebellum or cerebellum and spinal cord in the 100% of the piglets with CT, and APPV was not detected in any tissue sample from clinically non-affected piglets with the exception of the cerebellum of one piglet from Farm A. Piglets with CT had an odds ratio of 99.0 (95% CI 3.4, 2823.8; p = .0072) compared to piglets without CT to test positive for APPV by qRT-PCR. A subset of positive samples was selected for sequencing of the NS3 gene. Phylogenetic analysis revealed that Brazilian sequences of the NS3 formed an independent cluster and had the highest sequence identity with a sequence from the United States. This is the first identification of APPV infection in piglets with CT in South America.

Identification of a natural recombinant transmissible gastroenteritis virus between Purdue and Miller clusters in China.

Transmissible gastroenteritis virus (TGEV) is an infective coronavirus (CoV) that causes diarrhea-related morbidity and mortality in piglets. For the first time, a natural recombination strain of a TGEV Anhui Hefei (AHHF) virus between the Purdue and the Miller clusters was isolated from the small intestine content of piglets in China. A phylogenetic tree based on a complete genome sequence placed the TGEV AHHF strain between the Purdue and the Miller clusters. The results of a computational analysis of recombination showed that the TGEV AHHF strain is a natural recombinant strain between these clusters. Two breakpoints located in the open reading frame 1a (ORF1a) and spike (S) genes were identified. The pathogenicity of the TGEV AHHF strain was evaluated in piglets, and the results show that TGEV AHHF is an enteric pathogenic strain. These results provide valuable information about the recombination and evolution of CoVs and will facilitate future investigations of the molecular pathogenesis of TGEV.

Identification of bovine viral diarrhea virus type 2 in Korean native goat (Capra hircus).

In the genus Pestivirus, four genetically distinct viral species are currently recognized: bovine viral diarrhea viruses type 1 and 2 (BVDV-1, BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). BVDV-1 and BDV infections have been described in goat species. Since 1998, border disease (BD) like symptoms in goats have been reported repeatedly in two southern-most provinces of Korea, which until then had been regarded as being free from BD. As a result of retrospective investigations of BD-like syndrome in goat reported between 1998 and 2004, a pestivirus was identified from intestinal content of an affected kid submitted in 1999. Both sequences of 5'-non-coding region and complete N(pro) gene from the isolate were analyzed to identify the genotype. Interestingly, the results revealed that the isolate belonged to BVDV-2 that is rarely reported even in cattle. The isolate showed close relationship to North American and European strains rather than the geographically closer Japanese strains. To authors' knowledge, this is the first identification of BVDV-2 in goat species.

Feline immunodeficiency virus is concentrated in milk early in lactation.

We studied mother-to-offspring transmission of feline immunodeficiency virus (FIV), focusing on milk-borne virus transmission in order to assess its similarities to perinatal HIV transmission. We also attempted to evaluate the influence of intragestational treatment with 9-[2-(phosphono-methoxy)-propyl]adenine (PMPA) on virus transmission to offspring. Eleven female cats (queens), chronically infected with FIV-B-2542 and bred to an FIV-negative male, produced a total of 25 viable and 18 nonviable term kittens. Overall, the vertical transmission rate by untreated queens was 22%, similar to that for HIV, which unfortunately precluded adequate assessment of PMPA efficacy. However, at delivery 9 of 10 queens (90%) had higher viral RNA loads in milk (4 x 10(4) to 4 x 10(8) viral copies/ml) than in plasma (5 x 10(3) to 2.5 x 10(6) viral copies/ml). Conversely, 10 of 11 queens (91%) had lower proviral loads in milk cells (0 to 10(2) proviral copies/microg DNA) than blood cells (10(2) to 10(4) proviral copies/microg DNA). Thus, FIV is concentrated in early milk despite relatively low proviral loads in milk cells, suggesting that virus may be actively secreted by the mammary gland for dissemination to offspring. FIV provides a model for the study of milk-borne lentivirus transmission and assessment of strategies to reduce postnatal HIV vertical transmission.

Virulence of variola viruses for suckling mice.

We report work done in 1971 to determine the quantitative virulence for suckling mice of 26 variola virus isolates from different countries and from cases of differing severity. Strains of recognized variola major and variola minor viruses differed up to 100-fold (expressed as the harmonic mean dose of inoculum which killed mice 2-4 d old, inoculated intracranially, in 5 d). Isolates from Indonesia and from East and West Africa gave intermediate values. Unlike tests on chick embryos, this test distinguished between African and Indonesian isolates.

Viral isolation from cases of epidemic neuropathy in Cuba.

OBJECTIVE: To investigate the possibility of a viral agent in the central nervous system of patients with epidemic neuropathy. DESIGN: Virus isolation attempts, in cell cultures and suckling mice, from cerebrospinal fluid (CSF) of neuropathy patients and controls undergoing lumbar puncture for unrelated reasons. Serologic studies in patients, contacts, and controls. SETTING: An epidemic of optic and peripheral neuropathy affected more than 50,000 people in Cuba in 1991 through 1993. Illness was associated with dietary limitations and increased physical demands accompanying the shortages of food and fuel experienced in Cuba since 1989. Most patients responded to parenteral vitamin therapy, and the epidemic began to subside when oral vitamin supplementation was begun for the entire Cuban population. RESULTS: Coxsackievirus A9 (five isolates) and a similar, less cytopathic virus (100 isolates) were recovered from 105 (84%) of 125 CSF specimens from neuropathy patients. The strains with light cytopathic effect were antigenically related to Coxsackieviruses A9 and B4 by cross-neutralization and immunoblotting assays. Virus persisted in CSF of some patients for 1 to 12 months. Cerebrospinal fluid from patients and both types of virus from cell culture produced illness, including complete posterior flaccid paralysis, in newborn mice, and virus was reisolated from the mice. Mouse tissues and sural nerve biopsy specimens from patients were stained by immunoperoxidase and colloidal gold techniques using hyperimmune rabbit antisera against the virus with light cytopathic effect. CONCLUSIONS: Coxsackievirus A9 or an antigenically related agent with a light cytopathic effect was present in CSF of 84% of 125 patients with epidemic neuropathy. The role of these agents, probably in combination with nutritional factors, in the pathophysiology of the disease requires further investigation.

Experimental inoculation of mice with trypsin-resistant and trypsin-sensitive avian reoviruses.

Groups of sucking Swiss albino mice were inoculated by the intracerebral (i.c.), intraperitoneal (i.p.) or oral route with a trypsin-sensitive avian reovirus (TR1) or a trypsin-resistant (R2) reovirus. The viruses caused a number of effects, the most severe occurring after i.c. inoculation and the least after oral inoculation. They included incoordination and tremors, oiliness of the hair, and retarded growth. Patterns of viral persistence in tissues were similar for the two viruses, with high titres in the brain on days 3 and 6 after i.c. or i.p. injection. Both viruses were still present in the brain 21 days after i.c. injection. No virus was found in any tissue when TR1 was given orally. All groups "seroconverted" except the one infected orally with TR1, but neutralization titres were low. The effects resembled those described for mammalian reoviruses in mice. The results indicate that, for short periods, wild mice may be capable of transmitting avian reoviruses between poultry flocks. Furthermore, in the production of monoclonal antibodies to avian reoviruses in mice, it is possible that pathological changes will occur.

Passage of equine herpesvirus-1 in suckling mouse brain enhances extraneural virus growth and subsequent hematogenous neuroinvasion.

Intracerebral inoculation of field-isolates as well as established strains of equine herpesvirus-1 (EHV-1) in suckling mice results in viral replication in neurons and glial cells and induces encephalitis. By intraperitoneal (i.p.) inoculation, no histological lesion was observed in the central nervous system (CNS) in suckling mice with the EHV-1 HH1 strain (HH1), whereas a neuroadapted variant (NHH1) produced by serial passage of HH1 in the mouse brain caused severe encephalomyelitis after i.p. inoculation. The purpose of this study was to determine the route of neuroinvasion after i.p. inoculation of NHH1 and to clarify the effects of the brain passage on viral neuroinvasion. NHH1, but not HH1, targeted splenic and pulmonary macrophages and omental fat cells on days 1 and 2 post-inoculation (p.i.). From days 1 to 3 p.i., cell-associated viremia was occurred in NHH1-infected mice, but not in HH1-infected mice. On day 4 p.i., viral antigen was detected in a few endothelial cells, perivascular glial cells and neurons in the CNS in NHH1-infected mice. The number of viral antigen-positive cells increased markedly after day 5 p.i. In contrast, no viral antigen-positive cell was detected in the CNS in HH1-infected mice, except for a few nerve cells in the thoracic cord on day 4 p.i. These results suggest that NHH1 neuroinvasion is hematogenous and is correlated with enhanced extraneural virus growth.

Identification of pestiviruses contaminating cell lines and fetal calf sera.

Three pestiviruses in fetal calf serum (FCS), a pestivirus in porcine ST cell line, and two pestiviruses in two of five bovine cell lines were detected by RT-PCR method employing panpestivirus primers selected from the 5'-non-coding region (5'-NCR). The 288 bp products were sequenced in both directions. To identify these pestiviruses, their nucleotide sequences and those of reference pestiviruses were used for construction of a dendrogram. Three viruses present in FCS and a virus present in the bovine RP-15 cell line were identified as bovine viral diarrhea virus I (BVDV-1). Bovine viral diarrhea virus 2 (BVDV-2) was identified in a batch of the bovine MDBK cell line. A pestivirus contaminating the porcine ST cell line was identified as a Border disease virus (BDV).